Humanized genetically-engineered autochthonous brain tumors compatible with use in immunocompetent rodents

Abstract

The present invention describes methods and systems for generating an immunocompetent rodent model for cancer, including brain cancer. The invention also describes the immunocompetent rodent model for cancer.

Description

HUMANIZED GENETICALLY-ENGINEERED AUTOCHTHONOUS BRAIN TUMORS COMPATIBLE WITH USE IN IMMUNOCOMPETENT RODENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 687,923, filed August 28, 2024, the entirety of which is hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable form named “065472_000907WOPT_sequencelisting.xml”, having a size in bytes of 11,047 bytes, and created on August 27, 2025. The information contained in this computer readable form is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0003] This invention relates to immunocompetent non-human animal models.BACKGROUND

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Currently, there are no brain tumor models of immunocompetent rodents that are both autochthonous and humanized in the state of the art. There are different humanized mouse models that are used mainly for preclinical studies. These models are significantly different than the invention described herein. They refer mainly to mice that have been engrafted with something from humans, e.g., patient-derived xenograft (PDX). For cancer drug testing, most humanized models are those mice with an immunocompromised immune system that are engrafted with human tumor tissue and / or human immune cells. Thus, there is a need in the art for brain tumor models of immunocompetent rodents.Page 1 of 474918-4489-0203 4065472-000907WOPTSUMMARY OF THE INVENTION

[0006] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0007] Various embodiments of the invention provide for a method of generating a nonhuman transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising:(a) a donor vector, comprising:(i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule, the one or more murinized human DNA molecule, and(ii) paired recombinase recognition sites; and(b) two recombinases specific to the paired recombinase recognition sites.

[0008] Various embodiments of the invention provide for a method of generating a nonhuman transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising:(a) a donor vector, comprising:(i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murine DNA molecule not expressed in a murine brain, the one or more murine DNA molecule not expressed in the murine brain, and(ii) paired recombinase recognition sites; and(b) two recombinases specific to the paired recombinase recognition sites.

[0009] Various embodiments of the invention provide for a method of generating a nonhuman transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising:(a) a donor vector, comprising:(i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being aPage 2 of 474918-4489-0203 4065472-000907WOPTtumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain, the one or more murinized human DNA molecule and the one or more murine DNA molecule not expressed in the murine brain, and(ii) paired recombinase recognition sites; and(b) two recombinases specific to the paired recombinase recognition sites.

[0010] In various embodiments, the two recombinases are provided by(i) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or(ii) two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, or(iii) one mRNA encoding the two recombinases, or(iv) two mRNA, the first mRNA encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second mRNA encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, or(v) one viral vector comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or(vi) two viral vectors, the first viral vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second viral vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, or(vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase, or(viii) two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.

[0011] In various embodiments, the tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combination thereof

[0012] In various embodiments, the murinized human DNA molecule is a humanspecific gene associated with human neural precursor cell proliferation during development. In various embodiments, the human DNA molecule is a gene that has undergone accelerated evolution or a gene that regulate human brain development. That is, human-specific genePage 3 of 474918-4489-0203 4065472-000907WOPTassociated with human neural precursor cell proliferation during development has been murinized.

[0013] In various embodiments, the murinized human DNA molecule is a murinized ARHGAP11B gene. In various embodiments, the murinized ARHGAP11B gene is a chimeric gene named Arhgapl laB. In various embodiments, Arhgapl laB comprises the sequence MWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNSLPHSV VPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACLSSALPCDVA GLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMANPTVDILRYFFNFL KSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEKKGVYQTLSWKRYQPCWV LMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1). Thus, the murinized ARHGAP11B gene (Arhgapl laB) encodes SEQ ID NO:1. In various embodiments, the murinized human DNA molecule is a murinized NOTCH2NL gene, wherein the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB. Notch2nlB comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNGG TCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 2). Thus, the murinized NOTCH2NL gene (Notch2nlB) encodes SEQ ID NO:2. In various embodiments, the murinized human DNA molecule is murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.

[0014] In various embodiments, the murine DNA molecule not expressed in the murine brain is murine Notch2. In various embodiments, the murine Notch2 DNA molecule does not comprise its introns. In various embodiments, the murine Notch2 comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQPage 4 of 474918-4489-0203 4065472-000907WOPTDGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYA (SEQ ID N0:3). Thus, the murine Notch2 DNA molecule encodes SEQ ID NO:3).

[0015] In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain encodes SEQ ID NO:3 andPage 5 of 474918-4489-0203 4065472-000907WOPTSEQ ID NO:2. In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain further comprises a DNA molecule that encodes a linker and 2A peptides between SEQ ID NO:3 and SEQ ID NO:2. For example, it encodesMPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRGPage 6 of 474918-4489-0203 4065472-000907WOPTPDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYAGSGAT NFSLLKQAGDVEENPGPPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCV TYHNGTGFCRCPEGFLGEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGED CQYSTSHPCFVSRPCQNGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGST CTSVASQFSCKCPAGLTGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHC DSPYVPCAPSPCVNGGTCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 4).

[0016] In various embodiments, the donor vector is selected from the group consisting of plasmid, linear PCR, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC).

[0017] In various embodiments, the donor vector comprises at least four polyadenylation signals upstream from the transgene.

[0018] In various embodiments, the donor vector further comprises a post-transcriptional regulatory element. In various embodiments, the donor vector further comprises a polyadenylation signal downstream from the transgene. In various embodiments, the donor vector further comprises an open reading frame (ORF) that begins with a splice acceptor. In various embodiments, the donor vector further comprises a fluorescent reporter.

[0019] In various embodiments, the viral vector is an adeno-associated viral (AAV) vector. In various embodiments, the expression vector comprising recombinases are under tissuespecific promoters.

[0020] In various embodiments, the paired recombinase recognition sites are (i) loxP and flippase recognition target (FRT), and the recombinases are ere and flp, (ii) modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp, (ii) VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, or (iv) SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp.Page 7 of 474918-4489-0203 4065472-000907WOPT

[0021] In various embodiments, the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively.

[0022] In various embodiments, one or both of the paired recombinase recognition sites comprise a mutation.

[0023] In various embodiments, the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, or Hprt locus.

[0024] In various embodiments, the paired recombinase recognition sites comprises at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0025] In various embodiments, the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site. In various embodiments, the unidirectional recombinase recognition site is downstream to a promoter.

[0026] In various embodiments, the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

[0027] In various embodiments, the unidirectional recombinase is selected from Bxbl, Phic31, PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, WP, BL3, SPBc, K38, and any mutants thereof.

[0028] In various embodiments, the bidirectional recombinase is Flp.

[0029] In various embodiments, the unidirectional recombinase is Bxbl and the bidirectional recombinase is selected from Flp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof. In various embodiments, the unidirectional recombinase recognition site is attB or attP.

[0030] In various embodiments, the bidirectional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

[0031] In various embodiments, one or both of the recombinase recognition sites comprise a mutation.

[0032] In various embodiments, the donor vector comprises an intron, or part of an intron upstream and / or downstream from the transgene or nucleic acid encoding the RNA. In various embodiments, the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA.

[0033] Various embodiments provide for a non-human transgenic rodent, made by any one of the methods of the invention described herein.

[0034] In various embodiments, the transgenic rodent further carries a human-specific gene in its whole genome; for example, in a murinized form.

[0035] Various embodiments provide for a method of assessing the effects of a drug candidate, comprising: providing any one of the non-human rodent model of the inventionPage 8 of 474918-4489-0203 4065472-000907WOPTdescribed herein; administering the drug candidate to the non-human rodent model; and assessing the effects of the drug candidate on the non-human rodent model.

[0036] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0037] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0038] Figure 1 shows an exemplary genetic construct in accordance with various embodiments of the present invention. For example, cancer driver genes are positioned in the promoter-less first cistron whereas the human-specific murinized genes are placed in a secondary cistron. Bottom panel shows a murinized ARHGAP11B gene. It has both mouse and human coding sequences; the chimeric gene is named Arhgapl laB

[0039] Figure 2 shows additional exemplary genetic constructs in accordance with various embodiments of the present invention for comparing the human-specific murinized gene to a control. For example, both genetic constructs carry the same cancer driver genes positioned in the promoter-less first cistron differing in the secondary cistron. On the secondary cistron, one construct carries a human-specific murinized gene and blue fluorescent reporter transgene whereas the second construct carries only the blue fluorescent reporter transgene.

[0040] Figure 3 shows that immunocompetent mice with humanized brain tumors are more deadly. Bottom / left line: Survival curve of mice with humanized brain tumors. Top / Right line : Survival curve of mice with non-humanized brain tumors.

[0041] Figure 4 shows ARHGAP11B expression in human brain tumors. Bottom / left line: Survival curve of patients overexpressing ARHGAP11B. Top / right line : Survival curve of patients with low ARHGAP11B expression. Plot was generated using The Cancer Genome Atlas Program (TCGA) data.

[0042] Figure 5 shows additional exemplary genetic constructs in accordance with various embodiments of the present invention for comparing the functional full-length gene of NOTCH2NLB (N2NLB-FL) to a non-functional isoform (N2NLB-AEGF). E.g.: Both genetic constructs carry the same fluorescent reporter transgene and the cancer driver genes but differing in the NOTCH2NLB isoforms.

[0043] Figures 6A-6C show the effect of NOTCH2NLB isoforms in mouse brain tumors, data in homozygous mTFPl mice. MADR donor vectors include NOTCH2NLB isoformsPage 9 of 474918-4489-0203 4065472-000907WOPT(N2NLNLB-FL or N2NLB-AEGF), smFP_Myc_bright, mutant Pdgfra, mutant p53, and mutant Histone-3 (K27M). 6A: Coronal anterior section, close to the olfactory bulbs, 6B: Coronal section, close to the subventricular zone, 6C: Posterior coronal section, close to the Hippocampus. Images were taken 2 months post electroporation.

[0044] Figure 7A-7D shows a diagram of exemplary genetic constructs in accordance with various embodiments of the present invention for comparing murine genes and / or paralog human-specific murinized genes. 7A) A diagram showing a MADR donor vector carrying a reporter gene and the cancer driver genes positioned in the first cistron. The second cistron carries a murinized paralog human-specific control gene (Notch2nlB-Delta-EGF). 7B) A diagram showing a MADR donor vector carrying a reporter gene and the cancer driver genes positioned in the first cistron. The second cistron carries a murinized paralog human-specific gene (Notch2nlB- FL). 7C) A diagram showing a MADR donor vector carrying a reporter gene and the cancer driver genes positioned in the first cistron. The second cistron carries a murine gene (Notch2). 7D) A diagram showing a MADR donor vector carrying a reporter gene and the cancer driver genes positioned in the first cistron. The second cistron carries a murine gene (Notch2), a 2A sequence, and a murinized paralog human-specific gene (Notch2nlB-FL).

[0045] Figure 8 shows that immunocompetent mice with the murine Notch2 with or without the murinized human-specific Notch2nlB-FL are less deadly. Fine black line: Survival curve of mice with three cancer driver genes and Notch2nlB-Delta-EGF. Dashed line: Survival curve of mice with three cancer driver genes and Notch2nlB-FL. Grey line: Survival curve of mice with three cancer driver genes and Notch2. Thick black line: Survival curve of mice with three cancer driver genes, Notch2, and Notch2nlB-FL.DESCRIPTION OF THE INVENTION

[0046] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0047] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.Page 10 of 474918-4489-0203 4065472-000907WQPTIndeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0048] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0049] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0050] ‘Promoter-less vector” as used herein with respect to vectors, refers to a vector that does not contain a eukaryotic promoter.

[0051] In some embodiments, “control elements” refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present, so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0052] As used herein “paired” with respect to recombinase recognition sites refers to two recombinase recognition sites, one 5’ to a recited genetic element (e.g., gene of interest, promoter or other regulatory element) and one 3’ to the stated genetic element. Paired recombinase recognition sites may be identical (e.g., LoxP-LoxP), comprise a wild-type and a variant site (e.g., LoxP-Lox71 or the reverse), or sites of two different origins whether wild-type or variant. A variant of these sequences is any sequence that varies by one or more nucleotides and can be cleaved by its recombinase (e.g., Cre for Lox sites and Flippase for FRT sites). In certain embodiments, a variant LoxP site is paired with a wild-type or variant FRT site.Page 11 of 474918-4489-0203 4065472-000907WQPT

[0053] In certain embodiments, a variant FRT site is paired with a wild-type or variant LoxP site. In certain embodiments, a variant Lox selected from Lox71, Lox66, lox511, lox5171, lox2272 is paired with a wild-type or variant FRT site. In certain embodiments, a Lox71 site is paired with an FRT site or variant FRT site. In certain embodiments, a Lox66 site is paired with an FRT site or variant FRT site. In certain embodiments a variant FRT selected from FRT1, FRT2, FRT3, FRT4, FRT5, FRT12, FRT13, FRT14, FRT545 is paired with a wild-type FRT. In certain embodiments a variant FRT selected from FRT1, FRT2, FRT3, FRT4, FRT5, FRT12, FRT13, FRT14, FRT545 is paired with a wild-type LoxP. In certain embodiments, the choice of paired recombination sites increases the efficiency of transgenic insertion into a cellular genome by 25%, 50%, 75%, or 100% or more.

[0054] In some embodiments, “promoter region” is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 ’-direction) coding sequence.

[0055] In some embodiments, “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0056] ‘Murinized human gene” as used herein refers to human-specific genes that have elements that are murinized by using the paralogous rodent gene sequences, which are often duplicated, to create a chimeric protein that contains rodent proteome-derived peptides.

[0057] ‘Human-specific genes” are often associated with human neural precursor cell proliferation during development-a cell type / state often hijacked by tumors. Thus, genes which increase proliferation during development are likely to increase tumor precursor cell proliferation due to their neural precursor-like state.

[0058] ‘DNA molecule” as used herein include one or more coding and / or non-coding DNA sequences.

[0059] As used herein, a “subject” means a human or animal.

[0060] ‘Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. ThePage 12 of 474918-4489-0203 4065472-000907WOPTterm does not denote a particular age or sex. Thus adult and newborn subjects, as well as fetuses, whether male or female, are intended to be including within the scope of this term.

[0061] A “cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and / or all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastasis. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. As used herein, the term “invasive” refers to the ability to infiltrate and destroy surrounding tissue. In some embodiments, the tumor is a solid tumor.

[0062] Autochthonous mouse brain tumors induced by precise transgenesis, via the MADR technology, is shown to behave like their human counterparts. Although many same genes are expressed in both autochthonous mouse brain tumors and human brain tumors, there has not been a way that mouse tumors express human-specific genes. Thus, we decided to develop autochthonous mouse brain tumors with human-specific features by adding humanspecific genes that are potent for human brain evolution.

[0063] Because we are inducing endogenous brain tumors in immunocompetent mice, animals with “normal / regular” immune systems, we decided to engineer a chimeric gene that can avoid immune recognition but maintaining the function.

[0064] An example of this new genetically engineered gene has -80% mouse sequence and -20% human sequence, this can be considered as a “murinized” gene, an example is Arhgapl laB, (see e.g., figure 3).

[0065] Next, we compared validated autochthonous mouse brain tumors against humanized mouse brain tumors which differ only in the extra expression of the Arhgapl laB gene. As shown in figure 4, the mice with humanized mouse brain tumors died faster than those without the human-specific gene. A database search relating to human brain tumors show that patients who overexpress the ARHGAP11B gene tend to die faster than those patients that lower express the aforementioned human-specific gene, see figure 5.

[0066] Described herein we have engineered DNA cassettes in order to create humanized mouse brain tumors in immunocompetent mice. We have proved that humanized mouse brain tumors are more deadly than those tumors without human-specific genes. This development is a novel brain tumor model that is very important for preclinical drug testing. For example, different anti-cancer therapies that have a significant outcome in “classical” autochthonous mouse tumorsPage 13 of 474918-4489-0203 4065472-000907WQPTwill not have enough impact in the humanized mouse brain tumors, thus better drug candidates can be chosen before moving toward clinical trials.Methods of method of generating a non-human transgenic rodent

[0067] Various embodiments of the invention provide for a method of generating a non- human transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule, the one or more murinized human DNA molecule, and (ii) paired recombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0068] Various embodiments of the invention provide for a method of generating a non- human transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murine DNA molecule not expressed in a murine brain, the one or more murine DNA molecule not expressed in the murine brain, and (ii) paired recombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0069] Various embodiments of the invention provide for a method of generating a non- human transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain, the one or more murinized human DNA molecule and the one or more murine DNA molecule not expressed in the murine brain, and (ii) pairedPage 14 of 474918-4489-0203 4065472-000907WQPTrecombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0070] While the description herein refers to a “first” cistron and a “second” cistron, it is not intended to require that the first cistron is upstream to the second cistron. In some embodiments, the first cistron is upstream from the second cistron. In other embodiments, the second cistron is upstream from the first cistron.

[0071] Similarly, while the description herein refers to “first” and “second” recombinases, expression vectors, viral vectors, and the like, the designation is for convenience such as to provide antecedent basis and not to limit the features as a first or a second in order.

[0072] In various embodiments, the two recombinases are provided as proteins.

[0073] In various embodiments, the two recombinases are provided by one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites. In various embodiments, the two recombinases are provided by two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0074] In various embodiments, the two recombinases are provided by one mRNA encoding the two recombinases. In various embodiments, the two recombinases are provided by two mRNA, the first mRNA encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second mRNA encoding a second recombinase that is specific to the other of the paired recombinase recognition sites. In various embodiments, mRNA encoding a recombinase is delivered utilizing lipid nanoparticles, polymeric nanoparticles, viral vectors, or viral-like particles.

[0075] In various embodiments, the two recombinases are provided by one viral vector comprising two genes encoding recombinases specific to the paired recombinase recognition sites. In various embodiments, the two recombinases are provided by two viral vectors, the first viral vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second viral vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0076] In various embodiments, the two recombinases are provided by one recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase. In various embodiments, the two recombinases are provided by two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.Page 15 of 474918-4489-0203 4065472-000907WQPT

[0077] In various embodiments, the tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combination thereof. In various embodiments, the tumor driving gene is Pdgfr a, p53, and Histone 3. Additional examples of tumor driving genes include but are not limited to Egfrviii, Yapl-Mamldl, and Zfta-Rela.

[0078] In various embodiments, the murinized human DNA molecule is a humanspecific gene associated with human neural precursor cell proliferation during development. Examples include but are not limited to ARHGAP11B, NOTCH2NL, TEMEM14B, SRGAP2B, SRGAP2C, and TBC1D3. HAR1A is an additional example. That is, human-specific gene associated with human neural precursor cell proliferation during development has been murinized.

[0079] In various embodiments, the human DNA molecule is a gene that has undergone accelerated evolution or a gene that regulate human brain development. Examples include but are not limited to TKTL1 and TMEM161B. Thus, a murinized form of these are included in various embodiments of the present invention.

[0080] In various embodiments, the murinized human DNA molecule is a murinized ARHGAP11B gene.

[0081] In various embodiments, the murinized ARHGAP11B gene is a chimeric gene is named ArhgapllaB. In various embodiments, Arhgapl laB comprises the sequence: MWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNSLPHSV VPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACLSSALPCDVA GLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMANPTVDILRYFFNFL KSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEKKGVYQTLSWKRYQPCWV LMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1). Thus, the murinized ARHGAP11B gene (Arhgapl laB) encodes SEQ ID NO:1.

[0082] In various embodiments, the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB. In various embodiments, Notch2nlB comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNGG TCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO:2). Thus, murinized NOTCH2NL gene (Notch2nlB) encodes SEQ ID NO: 2.

[0083] In various embodiments, the murinized human DNA molecule is murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.Page 16 of 474918-4489-0203 4065472-000907WOPT

[0084] In various embodiments, the murine DNA molecule not expressed in the murine brain is murine Notch2. In various embodiments, the murine Notch2 DNA molecule does not comprise its introns. In various embodiments, the murine Notch2 comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLAPage 17 of 474918-4489-0203 4065472-000907WOPTARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYA (SEQ ID N0:3). Thus, the murine Notch2 DNA molecule encodes SEQ ID NO:3.

[0085] In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain encodes SEQ ID NO:3 and SEQ ID NO:2. In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain further comprises a DNA molecule that encodes a linker and 2A peptides between SEQ ID NO:3 and SEQ ID NO:2. For example, it encodesMPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGKPage 18 of 474918-4489-0203 4065472-000907WQPTNCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYAGAGAT NFSLLKQAGDVEENPGPPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTC VTYHNGTGFCRCPEGFLGEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGE DCQYSTSHPCFVSRPCQNGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGS TCTSVASQFSCKCPAGLTGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQH CDSPYVPCAPSPCVNGGTCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 4). GSG as bolded and italicized is the linker and ATNFSLLKQAGDVEENPGP (SEQ ID NO: 5), as bolded is the 2A sequence.

[0086] In various embodiments, the donor vector is selected from the group consisting of plasmid, linear PCR, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC). In various embodiments, the viral vector is an adeno-associated viral (AAV) vector.Page 19 of 474918-4489-0203 4065472-000907WQPT

[0087] In various embodiments, the donor vector comprises at least four polyadenylation signals upstream from the transgene. In various embodiments, the donor vector comprises 2, 3, 4, 5 or 6 polyadenylation signals upstream from the transgene.

[0088] In various embodiments, the donor vector further comprises a post-transcriptional regulatory element. In various embodiments, the donor vector further comprises a polyadenylation signal downstream from the transgene. In various embodiments, the donor vector further comprises an open reading frame (ORF) that begins with a splice acceptor. In various embodiments, the donor vector further comprises a fluorescent reporter.

[0089] Examples of fluorescent reporter proteins include but are not limited to EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T- Sapphire, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyanl, Midori-Ishi Cyan, TagCFP, mTFPl (Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, 12dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed- Express (Tl), DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFPl, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143. Thus, the donor vector can include a nucleic acid sequence encoding for any one or more of these fluorescent reporter proteins.

[0090] In various embodiments, the expression vector comprising nucleic acid sequence(s) encoding recombinases are under tissue-specific promoters. Examples include but are not limited to Gfap, Glast, and Synapsin.

[0091] In various embodiments, the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp, modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp, VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, or SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp. In various embodiments, the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively.

[0092] In various embodiments, one or both of the paired recombinase recognition sites comprise a mutation. In various embodiments, the mutation for loxP is selected from lox71, lox75, lox44, loxJT15, loxJT12, loxJT510, lox66, lox76, lox43, loxJTZ2, loxJTZ17, loxKR3, loxBait, lox5171, lox2272, lox2722, m2, and combinations thereof. In various embodiments, the mutation for FRT is selected from FRT+10, FRT+11, FRT-10, FRT-11, F3, F5, F13, F14, F15, F5T2, F545, f2161, f2151, f2262, f61, and combinations thereof. The mutation can allow for better transgenesis, and thus, new transgenic mice do not need to be generated. Furthermore,Page 20 of 474918-4489-0203 4065472-000907WOPTcombinatorial experiments can be applied in a shorter window of time which allows for results to be obtained immediately when more than two different donor plasmids are used. This is also valuable in models wherein the organisms develop faster than mice.

[0093] In various embodiments, the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, or Hprt locus.

[0094] In various embodiments, the paired recombinase recognition sites comprises at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0095] In various embodiments, the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site. In various embodiments, the unidirectional recombinase recognition site is downstream to a promoter. Examples of promoters include but are not limited to U6, Cag, CMV and EFla.

[0096] In various embodiments, the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

[0097] In various embodiments, the unidirectional recombinase is selected from Bxbl, Phic31, PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, WP, BL3, SPBc, K38, and any mutants thereof. In various embodiments, the bidirectional recombinase is Flp. In various embodiments, the unidirectional recombinase is Bxbl and the bidirectional recombinase is selected from Flp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof. In various embodiments, the unidirectional recombinase recognition site is attB or attP. In various embodiments, the bidirectional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

[0098] In various embodiments, one or both of the recombinase recognition sites comprise a mutation. In various embodiments, the mutation for loxP is selected from lox71, lox75, lox44, loxJT15, loxJT12, loxJT510, lox66, lox76, lox43, loxJTZ2, loxJTZ17, loxKR3, loxBait, lox5171, lox2272, lox2722, m2, and combinations thereof. In various embodiments, the mutation for FRT is selected from FRT+10, FRT+11, FRT-10, FRT-11, F3, F5, F13, F14, F15, F5T2, F545, f2161, f2151 , f2262, f61, and combinations thereof.

[0099] In various embodiments, the donor vector comprises an intron, or part of an intron upstream and / or downstream from the transgene.

[0100] In various embodiments, the transgene is a nucleic acid encoding an RNA. In various embodiments, the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA.Page 21 of 474918-4489-0203 4065472-000907WQPT

[0101] In various embodiments, the transgenic rodent is an immunocompetent transgenic rodent. In various embodiments, the transgenic rodent is a mouse (e.g., immunocompetent mouse). In various embodiments, the transgenic rodent is a rat (e.g., immunocompetent rat).Systems for generating a non-human transgenic rodent

[0102] Various embodiments provide for a system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule, the one or more murinized human DNA molecule, and (ii) paired recombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0103] Various embodiments provide for a system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murine DNA molecule not expressed in a murine brain, the one or more murine DNA molecule not expressed in the murine brain, and (ii) paired recombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0104] Various embodiments provide for a system comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain, the one or more murinized human DNA molecule and the one or more murine DNA molecule not expressed in the murine brain, and (ii) paired recombinase recognition sites; and (b) two recombinases specific to the paired recombinase recognition sites.

[0105] While the description herein refers to a “first” cistron and a “second” cistron, it is not intended to require that the first cistron is upstream to the second cistron. In some embodiments, the first cistron is upstream from the second cistron. In other embodiments, the second cistron is upstream from the first cistron.

[0106] Similarly, while the description herein refers to “first” and “second” recombinases, expression vectors, viral vectors, and the like, the designation is for convenience such as to provide antecedent basis and not to limit the features a first or a second in order.Page 22 of 474918-4489-0203 4065472-000907WOPT

[0107] In various embodiments, the two recombinases are provided as proteins.

[0108] In various embodiments, the two recombinases are provided by one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites. In various embodiments, the two recombinases are provided by two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0109] In various embodiments, the two recombinases are provided by one mRNA encoding the two recombinases. In various embodiments, the two recombinases are provided by two mRNA, the first mRNA encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second mRNA encoding a second recombinase that is specific to the other of the paired recombinase recognition sites. In various embodiments, mRNA encoding a recombinase is delivered utilizing lipid nanoparticles, polymeric nanoparticles, viral vectors, or viral-like particles.

[0110] In various embodiments, the two recombinases are provided by one viral vector comprising two genes encoding recombinases specific to the paired recombinase recognition sites. In various embodiments, the two recombinases are provided by two viral vectors, the first viral vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second viral vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.[OHl] In various embodiments, the two recombinases are provided by one recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase. In various embodiments, the two recombinases are provided by two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.

[0112] In various embodiments, the tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combination thereof. In various embodiments, the tumor driving gene is Pdgfr a, p53, and Histone 3. Additional examples of tumor driving genes include but are not limited to Egfrviii, Yapl-Mamldl, and Zfta-Rela.

[0113] In various embodiments, the murinized human DNA molecule is a humanspecific gene associated with human neural precursor cell proliferation during development. Examples include but are not limited to ARHGAP11B, NOTCH2NL, TEMEM14B, SRGAP2B, SRGAP2C, and TBC1D3. HAR1A is an additional example. That is, human-specific genePage 23 of 474918-4489-0203 4065472-000907WOPTassociated with human neural precursor cell proliferation during development has been murinized.

[0114] In various embodiments, the human DNA molecule is a gene that has undergone accelerated evolution or a gene that regulate human brain development. Examples include but are not limited to TKTL1 and TMEM161B. Thus, murinized form of these genes are included in various embodiments of the invention.

[0115] In various embodiments, the murinized human DNA molecule is a murinized ARHGAP11B gene. In various embodiments, the murinized ARHGAP11B gene is a chimeric gene is named ArhgapllaB. In various embodiments, Arhgapl laB comprises the sequence: MWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNSLPHSV VPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACLSSALPCDVA GLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMANPTVDILRYFFNFL KSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEKKGVYQTLSWKRYQPCWV LMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1). Thus, the murinized ARHGAP11B gene (Arhgapl laB) encodes SEQ ID NO:1.

[0116] In various embodiments, the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB. In various embodiments, Notch2nlB comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNGG TCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO:2). Thus, murinized NOTCH2NL gene (Notch2nlB) encodes SEQ ID NO: 2.

[0117] In various embodiments, the murinized human DNA molecule is murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.

[0118] In various embodiments, the murine DNA molecule not expressed in the murine brain is murine Notch2. In various embodiments, the murine Notch2 DNA molecule does not comprise its introns. In various embodiments, the murine Notch2 comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKGPage 24 of 474918-4489-0203 4065472-000907WQPTADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQDGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQPKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTPPage 25 of 474918-4489-0203 4065472-000907WQPTSPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYA (SEQ ID N0:3). Thus, the murine Notch2 DNA molecule encodes SEQ ID NO:3).

[0119] In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain encodes SEQ ID NO:3 and SEQ ID NO:2. In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain further comprises a DNA molecule that encodes a linker and 2A peptides between SEQ ID NO:3 and SEQ ID NO:2. For example, it encodesMPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNIDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALMPage 26 of 474918-4489-0203 4065472-000907WQPTVYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYAGSGAT NFSLLKQAGDVEENPGPPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCV TYHNGTGFCRCPEGFLGEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGED CQYSTSHPCFVSRPCQNGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGST CTSVASQFSCKCPAGLTGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHC DSPYVPCAPSPCVNGGTCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 4).

[0120] In various embodiments, the donor vector is selected from the group consisting of plasmid, linear PCR, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC). In various embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0121] In various embodiments, the donor vector comprises at least four polyadenylation signals upstream from the transgene. In various embodiments, the donor vector comprises 2, 3, 4, 5 or 6 polyadenylation signals upstream from the transgene.

[0122] In various embodiments, the donor vector further comprises a post-transcriptional regulatory element. In various embodiments, the donor vector further comprises a polyadenylation signal downstream from the transgene. In various embodiments, the donor vector further comprises an open reading frame (ORF) that begins with a splice acceptor. In various embodiments, the donor vector further comprises a fluorescent reporter.

[0123] Examples of fluorescent reporter proteins include but are not limited to EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, T-Page 27 of 474918-4489-0203 4065472-000907WQPTSapphire, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyanl, Midori-Ishi Cyan, TagCFP, mTFPl (Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, 12dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed- Express (Tl), DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFPl, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143. Thus, the donor vector can include a nucleic acid sequence encoding for any one or more of these fluorescent reporter proteins.

[0124] In various embodiments, the expression vector comprising nucleic acid sequence(s) encoding recombinases are under tissue-specific promoters. Examples include but are not limited to Gfap, Glast, and Synapsin.

[0125] In various embodiments, the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp, modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp, VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, or SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp. In various embodiments, the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively.

[0126] In various embodiments, one or both of the paired recombinase recognition sites comprise a mutation. In various embodiments, the mutation for loxP is selected from lox71, lox75, lox44, loxJT15, loxJT12, loxJT510, lox66, lox76, lox43, loxJTZ2, loxJTZ17, loxKR3, loxBait, lox5171, lox2272, lox2722, m2, and combinations thereof. In various embodiments, the mutation for FRT is selected from FRT+10, FRT+11, FRT-10, FRT-11, F3, F5, F13, F14, F15, F5T2, F545, f2161, f2151, f2262, f61, and combinations thereof. The mutation can allow for better transgenesis, and thus, new transgenic mice do not need to be generated. Furthermore, combinatorial experiments can be applied in a shorter window of time which allows for results to be obtained immediately when more than two different donor plasmids are used. This is also valuable in models wherein the organisms develop faster than mice.

[0127] In various embodiments, the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, or Hprt locus.

[0128] In various embodiments, the paired recombinase recognition sites comprises at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0129] In various embodiments, the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site. In various embodiments, thePage 28 of 474918-4489-0203 4065472-000907WQPTunidirectional recombinase recognition site is downstream to a promoter. Examples of promoters include but are not limited to U6, Cag, CMV and EFla.

[0130] In various embodiments, the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

[0131] In various embodiments, the unidirectional recombinase is selected from Bxbl, Phic31, PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, WP, BL3, SPBc, K38, and any mutants thereof. In various embodiments, the bidirectional recombinase is Flp. In various embodiments, the unidirectional recombinase is Bxbl and the bidirectional recombinase is selected from Flp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof. In various embodiments, the unidirectional recombinase recognition site is attB or attP. In various embodiments, the bidirectional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

[0132] In various embodiments, one or both of the recombinase recognition sites comprise a mutation. In various embodiments, the mutation for loxP is selected from lox71, lox75, lox44, loxJT15, loxJT12, loxJT510, lox66, lox76, lox43, loxJTZ2, loxJTZ17, loxKR3, loxBait, lox5171, lox2272, lox2722, m2, and combinations thereof. In various embodiments, the mutation for FRT is selected from FRT+10, FRT+11, FRT-10, FRT-11, F3, F5, F13, F14, F15, F5T2, F545, f2161, f2151 , f2262, f61, and combinations thereof.

[0133] In various embodiments, the donor vector comprises an intron, or part of an intron upstream and / or downstream from the transgene.

[0134] In various embodiments, the transgene is a nucleic acid encoding an RNA. In various embodiments, the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA.

[0135] In various embodiments, the transgenic rodent is an immunocompetent transgenic rodent. In various embodiments, the transgenic rodent is a mouse (e.g., immunocompetent mouse). In various embodiments, the transgenic rodent is a rat (e.g., immunocompetent rat).Non-human transgenic rodents

[0136] Various embodiments provide for a non-human transgenic rodent, made by any one of the methods of the present invention as described herein. In various embodiments, the transgenic rodent carries a human-specific gene in its whole genome. In various embodiments, the human-specific gene is in a murinized form.

[0137] Thus in some embodiments, the non-human transgenic rodent carries a tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combinationPage 29 of 474918-4489-0203 4065472-000907WQPTthereof. In various embodiments, the non-human transgenic rodent carries Pdgfr a, p53, and Histone 3. In various embodiments, the non-human transgenic rodent carries Egfrviii, Yapl- Mamldl, Zfta-Rela or a combination thereof.

[0138] In some embodiments, the non-human transgenic rodent further carries a humanspecific gene associated with human neural precursor cell proliferation during development. Examples include but are not limited to ARHGAP11B, NOTCH2NL, TEMEM14B, SRGAP2B, SRGAP2C, and TBC1D3. HAR1A is an additional example. In various embodiments, the human-specific gene is in a murinized form.

[0139] In some embodiments, the non-human transgenic rodent further carries a gene that has undergone accelerated evolution or a gene that regulate human brain development. Examples include but are not limited to TKTL1 and TMEM161B. In various embodiments, the gene that has undergone accelerated evolution or a gene that regulate human brain development is in a murinized form.

[0140] In some embodiments, the non-human transgenic rodent further carries a murinized ARHGAP1 IB gene.

[0141] In various embodiments, the murinized ARHGAP11B gene is a chimeric gene named ArhgapllaB. In various embodiments, Arhgapl laB comprises the sequence: MWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNSLPHSV VPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACLSSALPCDVA GLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMANPTVDILRYFFNFL KSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEKKGVYQTLSWKRYQPCWV LMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1). Thus, the murinized ARHGAP11B gene (Arhgapl laB) encodes SEQ ID NO:1.

[0142] In some embodiments, the non-human transgenic rodent further carries a murinized NOTCH2NL gene. In some embodiments, the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB. In various embodiments, Notch2nlB comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNGG TCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO:2). Thus, murinized NOTCH2NL gene (Notch2nlB) encodes SEQ ID NO: 2.

[0143] In some embodiments, the non-human transgenic rodent carries murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.Page 30 of 474918-4489-0203 4065472-000907WQPT

[0144] In some embodiments, the non-human transgenic rodent carries a murine DNA molecule not expressed in a murine brain. In some embodiments, the non-human transgenic rodent carries murine Notch2. In various embodiments, the murine Notch2 DNA molecule does not comprise its introns. In various embodiments, the murine Notch2 comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRGPage 31 of 474918-4489-0203 4065472-000907WOPTPDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYA (SEQ ID N0:3). Thus, the murine Notch2 DNA molecule encodes SEQ ID NO:3.

[0145] In some embodiments, the non-human transgenic rodent carries one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain. In some embodiments, one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain encodes SEQ ID NO: 3 and SEQ ID NO:2. In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain further comprises a DNA molecule that encodes a linker and 2A peptides between SEQ ID NO:3 and SEQ ID NO:2. For example, it encodesMPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSNPCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNFDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISKPage 32 of 474918-4489-0203 4065472-000907WQPTPCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGK NCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYAGSGAT NFSLLKQAGDVEENPGPPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCV TYHNGTGFCRCPEGFLGEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGED CQYSTSHPCFVSRPCQNGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGST CTSVASQFSCKCPAGLTGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHC DSPYVPCAPSPCVNGGTCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 4).Page 33 of 474918-4489-0203 4065472-000907WQPT

[0146] In various embodiments, the transgenic rodent is an immunocompetent transgenic rodent. In various embodiments, the transgenic rodent is a mouse (e.g., immunocompetent mouse). In various embodiments, the transgenic rodent is a rat (e.g., immunocompetent rat).Drug screening

[0147] Various embodiments provide for a method of assessing the effects of a drug candidate, comprising: providing any one of the non-human rodent model of the present invention; administering the drug candidate to the non-human rodent model; and assessing the effects of the drug candidate on the non-human rodent model. Effects accessed can include but are not limited to cell viability, tumoroids viability, dose-response relationship, transcriptional changes, and epigenetic changes.

[0148] In some embodiments, the non-human transgenic rodent carries a tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combination thereof. In various embodiments, the non-human transgenic rodent carries Pdgfr a, p53, and Histone 3. In various embodiments, the non-human transgenic rodent carries Egfrviii, Yapl-Mamldl, Zfta- Rela or a combination thereof.

[0149] In some embodiments, the non-human transgenic rodent further carries a humanspecific gene associated with human neural precursor cell proliferation during development. Examples include but are not limited to ARHGAP11B, NOTCH2NL, TEMEM14B, SRGAP2B, SRGAP2C, and TBC1D3. HAR1A is an additional example. In various embodiments, humanspecific gene associated with human neural precursor cell proliferation during development is in a murinized form.

[0150] In some embodiments, the non-human transgenic rodent further carries a gene that has undergone accelerated evolution or a gene that regulate human brain development. Examples include but are not limited to TKTL1 and TMEM161B. In various embodiments, a gene that has undergone accelerated evolution or a gene that regulate human brain development is in a murinized form.

[0151] In some embodiments, the non-human transgenic rodent further carries a murinized ARHGAP11B gene. In various embodiments, the murinized ARHGAP11B gene is a chimeric gene named Arhgapl laB. In various embodiments, ArhgapllaB comprises the sequence: MWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNSLPHSV VPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACLSSALPCDVA GLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMANPTVDILRYFFNFL KSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEKKGVYQTLSWKRYQPCWVPage 34 of 474918-4489-0203 4065472-000907WOPTLMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1). Thus, the murmized ARHGAP11B gene (Arhgapl laB) encodes SEQ ID NO:1.

[0152] In some embodiments, the non-human transgenic rodent further carries a murinized NOTCH2NL gene. In some embodiments, the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB. In various embodiments, Notch2nlB comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNGG TCRQTGDFTFECNCLPETVRRGTELWERDREVWNGKEHDEN (SEQ ID NO: 2). Thus, the murinized NOTCH2NL gene (Notch2nlB) encodes SEQ ID NO: 2.

[0153] In some embodiments, the non-human transgenic rodent carries murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.

[0154] In some embodiments, the non-human transgenic rodent carries a murine DNA molecule not expressed in a murine brain. In some embodiments, the non-human transgenic rodent carries murine Notch2. In various embodiments, the murine Notch2 does not comprise its introns. In various embodiments, the murine Notch2 comprises the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCRCPEGFL GEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYSTSHPCFVSRPCQ NGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTCTSVASQFSCKCPAGL TGQKCEADINECDIPGRCQHGGTCLNLPGSYRCQCPQGFTGQHCDSPYVPCAPSPCVNG GTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHKCQNGGVCVDGVNTYNCRCPPQWT GQFCTEDVDECLLQPNACQNGGTCTNRNGGYGCVCVNGWSGDDCSENIDDCAYASCTP GSTCIDRVASFSCLCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYICTCPQGYKG ADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQ NDATCLDKIGGFTCLCMPGFKGVHCELEVNECQSNPCVNNGQCVDKVNRFQCLCPPGFT GPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGILCDENIDNCDPDPCHHGQCQ DGIDSYTCICNPGYMGAICSDQIDECYSSPCLNDGRCIDLVNGYQCNCQPGTSGLNCEINF DDCASNPCMHGVCVDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINDVNGFR CICPEGPHHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGVNCEVDKNECLSN PCQNGGTCNNLVNGYRCTCKKGFKGYNCQVNIDECASNPCLNQGTCFDDVSGYTCHC MLPYTGKNCQTVLAPCSPNPCENAAVCKEAPNFESFSCLCAPGWQGKRCTVDVDECISK PCMNNGVCHNTQGSYVCECPPGFSGMDCEEDINDCLANPCQNGGSCVDHVNTFSCQCH PGFIGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCTCPAGFHGVHCENNIDECTESSCFN GGTCVDGINSFSCLCPVGFTGPFCLHDINECSSNPCLNAGTCVDGLGTYRCICPLGYTGKPage 35 of 474918-4489-0203 4065472-000907WQPTNCQTLVNLCSRSPCKNKGTCVQEKARPHCLCPPGWDGAYCDVLNVSCKAAALQKGVP VEHLCQHSGICINAGNTHHCQCPLGYTGSYCEEQLDECASNPCQHGATCNDFIGGYRCE CVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGLLCEENIDECAGGP HCLNGGQCVDRIGGYTCRCLPGFAGERCEGDINECLSNPCSSEGSLDCVQLKNNYNCICR SAFTGRHCETFLDVCPQKPCLNGGTCAVASNMPDGFICRCPPGFSGARCQSSCGQVKCR RGEQCIHTDSGPRCFCLNPKDCESGCASNPCQHGGTCYPQRQPPHYSCRCPPSFGGSHCE LYTAPTSTPPATCQSQYCADKARDGICDEACNSHACQWDGGDCSLTMEDPWANCTSTL RCWEYINNQCDEQCNTAECLFDNFECQRNSKTCKYDKYCADHFKDNHCDQGCNSEECG WDGLDCASDQPENLAEGTLIIVVLLPPEQLLQDSRSFLRALGTLLHTNLRIKQDSQGALM VYPYFGEKSAAMKKQKMTRRSLPEEQEQEQEVIGSKIFLEIDNRQCVQDSDQCFKNTDA AAALLASHAIQGTLSYPLVSVFSELESPRNAQLLYLLAVAWIILFFILLGVIMAKRKRKH GFLWLPEGFTLRRDSSNHKRREPVGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQ PKKAKAEDEALLSEDDPIDRRPWTQQHLEAADIRHTPSLALTPPQAEQEVDVLDVNVRG PDGCTPLMLASLRGGSSDLSDEDEDAEDSSANIITDLVYQGASLQAQTDRTGEMALHLA ARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARM NDGTTPLILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLK NGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH DIVRLLDEYNVTPSPPGTVLTSALSPVLCGPNRSFLSLKHTPMGKKARRPNTKSTMPTSLP NLAKEAKDAKGSRRKKCLNEKVQLSESSVTLSPVDSLESPHTYVSDATSSPMITSPGILQA SPTPLLAAAAPAAPVHTQHALSFSNLHDMQPLAPGASTVLPSVSQLLSHHHIAPPGSSSA GSLGRLHPVPVPADWMNRVEMNETQYSEMFGMVLAPAEGAHPGIAAPQSRPPEGKHMS TQREPLPPIVTFQLIPKGSIAQAAGAPQTQSSCPPAVAGPLPSMYQIPEMPRLPSVAFPPTM MPQQEGQVAQTIVPTYHPFPASVGKYPTPPSQHSYASSNAAERTPSHGGHLQGEHPYLTP SPESPDQWSSSSPHSASDWSDVTTSPTPGGGGGGQRGPGTHMSEPPHSNMQVYA (SEQ ID NO:3). Thus, the murine Notch2 encodes SEQ ID NO:3).

[0155] In some embodiments, the non-human transgenic rodent carries one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain. In some embodiments, one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain encodes SEQ ID NO: 3 and SEQ ID NO:2. In various embodiments, the one or more murinized human DNA molecule and one or more murine DNA molecule not expressed in a murine brain further comprises a DNA molecule that encodes a linker and 2A peptides between SEQ ID NO:3 and SEQ ID NO:2. For example, it encodes SEQ ID NO:4.Page 36 of 474918-4489-0203 4065472-000907WQPT

[0156] In various embodiments, the transgenic rodent is an immunocompetent transgenic rodent. In various embodiments, the transgenic rodent is a mouse (e.g., immunocompetent mouse). In various embodiments, the transgenic rodent is a rat (e.g., immunocompetent rat).EXAMPLES

[0157] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1 Experimental proceduresMice

[0158] All mice used were maintained and euthanized according to the Cedars-Sinai Institutional Animal Care and Use committee. mT / mG (Gt(ROSA)26Sortm4(ACTB-tdTomato,- EGFP)Luo / J) mice (Muzumdar, Tasic, Miyamichi, Li, & Luo, 2007) were bred with C57BL / 6J mice to generate heterozygous mice. Homozygous R26-CAG-LF-mTFPl were bred to get homozygous mice. Pregnant dams were kept in single cages and pups were kept with their mothers until P21, in the institutional animal facility under standard 12: 12 h light / dark cycles.Electroporation

[0159] Male and female pups between postnatal day (P) 2 and P3 were used for electroporation. P2-P3 pups were placed on ice for ~5 min. All DNA mixtures contained 0.5- Img / ml of Flp-Cre expression vector and the MADR donor plasmid. Fast green dye was added (10%v / v) to the mixture, which was injected into the lateral ventricle. Platinum Tweezertrodes delivered 5 pulses of 120 V (50ms; separated by 950 ms) from the ECM 830 System (Harvard Apparatus). SignaGel was applied to increase conductance. Mice were warmed under a heat lamp and returned to their cages.Plasmid cloning

[0160] The pDonor plasmids were derived from MADR-pDonors using NEBuilder HiFi DNA Assembly Master Mix (NEB) in combination with standard restriction digestion techniques (Kim et al., 2019). Briefly, specific recombination sites were created by oligo synthesis and inserted into the MADR-pDonor. Expression recombinase vectors were derived from pCag-FlpO- 2A-Cre EV (Addgene 129419) that were previously validated (Kim et al., 2019). Downstream generation of plasmids were done by removing the existing ORF and adding a new cassette using HiFi DNA Assembly. PCR was done using a standard protocol with KAPA HiFi PCR reagents.Page 37 of 474918-4489-0203 4065472-000907WQPTEpifluorescence microscopy

[0161] Images were taken with a fluorescence microscope (ECHO Revolve) using the respective fluorescence channels and filters.Example 2 ResultsChimeric ArhgapllaB expression induces a more deadly brain tumor

[0162] We chose to model pediatric glioblastoma multiforme where a variety of mutations occur. The strategy followed was to make MADR donor vectors with two different cistrons flanked by loxP and FRT sites, Figure 1. The first cistron carries a gene reporter that codifies a fluorescent protein that facilitates the visualization of the tumor. It also contains the cancer driver genes which are H3F3A (K27M) mutant, Pdgfra (D842V) mutant, and dominantnegative Trp53 (R270H). Besides, this is a promoter-less cistron. Then, in a secondary cistron, we added a murinized version of the human-specific gene, ARHGAP11B. Because the murinized gene contains coding sequences of both the mouse Arhgapl la gene and the human ARHGAP1 IB gene, the chimeric gene is named ArhgapllaB (Figure 1, bottom). Finally, the expression of the second cistron is under the mouse Arhgapl la promoter, Figure 1.

[0163] We decided to use MADR technology in heterozygous mTmG mice to generate single-copy in vivo tumors. Besides, two MADR vectors were made to compare the effect of Arhgapl laB. For this purpose, one MADR vector has a sequence that codifies for a blue fluorescent protein upstream to the Arhgapl laB gene in the second cistron. As a control, a second MADR vector was made where the secondary cistron doesn’t contain the chimeric Arhgapl laB, Figure 2.

[0164] Survival analysis exhibits that the mouse brain tumor that expresses the chimeric Arhgapl laB is more deadly compared to the control tumor which does not express Arhgapl IB, Figure 3. This data suggests that adding a human-specific feature to an endogenous mouse brain tumor can make it more aggressive, and likely to be a better brain tumor model. Data of human brain tumor data shows that high expression of the human-specific gene, ARHGAP11B, is more deadly compared to those tumors that have low expression of ARHGAP11B, Figure 4. This data shows the relevance of adding human-specific genes, expressed in human tumors, to autochthonous mouse brain tumors.Effect of the Human-specific NOTCH2NLB on the growth of mouse brain tumors

[0165] We decided to test NOTCH2NLB (N2NLB) which is another human-specific gene. N2NLB is a smaller and paralog gene of NOTCH2. For testing the effect of N2NLB we created two MADR donor vectors. One MADR vector carries the full-length sequence of N2NLBPage 38 of 474918-4489-0203 4065472-000907WQPT(N2NLB-FL) which is the functional gene. For the MADR control vector, we used a nonfunctional N2NLB which does not contain the EGF repeats (AEGF). Both MADR donor vectors carry a gene reporter that codifies a fluorescent protein, smFP Myc bright, that facilitates the visualization of the tumor, the cancer driver genes which are H3F3A (K27M) mutant, Pdgfra (D842V) mutant, and dominant-negative Trp53 (R270H), Figure 5. Homozygous mTFPl mice were used to generate single- and dual-copy in vivo tumors. Staining of TFP1 shows the correct Cre-loxP and FlpO-FRT recombination, whereas MYC-tag staining shows the expression of the MADR cassettes facilitating brain tumor visualization. Two months after electroporation, the tumor expressing the functional N2NLB-FL is smaller compared to the control in which a nonfunctional N2NLB is expressed, Figure 6A-C. This data shows the effect of the human-specific gene NOTCHN2NLB on mouse brain tumor growth.Example 3 ResultsThe effect of murine Notch2 with or without the murinized Notch2nlB-FL induces less deadly brain tumors

[0166] A pediatric glioblastoma multiforme which a variety of mutations occur, was modeled. Four MADR donor vectors were developed with two cistrons flanked by loxP and FRT sites, Figure 7. All four MADR donor vectors carry the same promoter-less first cistron. The first cistron contains three cancer driver genes, which are H3F3A (K27M) mutant fused to GFP, Pdgfra (D842V) mutant, and dominant-negative Trp53 (R270H). Then, in the secondary cistron, different isoforms of Notch2 were added under a constitutive promoter. The first MADR vector carries a murinized Notch2nlB-Delta-EGF, Figure 7A. The second MADR vector carries a murinized Notch2nlB-FL, Figure 7B. The third MADR vector carries a murine Notch2, Figure 7C. The fourth MADR vector carries a murine Notch2, a 2A sequence, and a murinized Notch2nlB-FL, Figure 7D. The MADR vectors were electroporated in heterozygous mTmG mice to generate single-copy in vivo tumors.

[0167] Survival analysis exhibits that the mouse brain tumors that express the murine Notch2 with or without the murinized Notch2nlB-FL are less deadly compared to both the nonactive isoform of the murinized Notch2nlB (Notch2nlB-DeltaEGF) and to the murinized full- length Notch2nlB (Notch2nlB-FL), Figure 8. This data shows the impact of expressing missed ortholog murine genes and murinized human-specific genes in autochthonous mouse brain tumors.Page 39 of 474918-4489-0203 4065472-000907WQPT

[0168] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0169] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0170] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0171] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be construed to cover both the singular and the plural. The recitation of ranges ofPage 40 of 474918-4489-0203 4065472-000907WOPTvalues herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0172] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0173] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.Page 41 of 474918-4489-0203 4065472-000907WQPT

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of generating a non-human transgenic rodent, comprising: inducing an autochthonous tumor in an immunocompetent rodent by mosaic analysis by dual recombinase cassette exchange (MADR) by delivering a system into cells of the immunocompetent rodent, the system comprising:(a) a donor vector, comprising:(i) one or more polyadenylation signals or transcription stop element upstream from a first cistron and a second cistron, the first cistron comprising a transgene, the transgene being a tumor driving gene, wherein the first cistron is promoter-less, the second cistron comprising a promoter for one or more murinized human DNA molecule, the one or more murinized human DNA molecule, and(ii) paired recombinase recognition sites; and(b) two recombinases specific to the paired recombinase recognition sites.

2. The method of claim 1, wherein the two recombinases are provided by(i) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or(ii) two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, or(iii) one mRNA encoding the two recombinases, or(iv) two mRNA, the first mRNA encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second mRNA encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, or(v) one viral vector comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or(vi) two viral vectors, the first viral vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second viral vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites, orPage 42 of 474918-4489-0203 4065472-000907WQPT(vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase, or(viii) two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.

3. The method of claim 1 or claim 2, wherein the tumor driving gene is platelet-derived growth factor a (Pdgfr a), p53, Histone 3 or a combination thereof4. The method of any one of claims 1-3, wherein the murinized human DNA molecule is a human-specific gene associated with human neural precursor cell proliferation during development.

5. The method of any one of claims 1-4, wherein the human DNA molecule is a gene that has undergone accelerated evolution or a gene that regulate human brain development.

6. The method of any one of claims 1-5, wherein the murinized human DNA molecule is a murinized ARHGAP1 IB gene.

7. The method of claim 6, wherein the murinized ARHGAP11B gene is a chimeric gene named Arhgapl laB.

8. The method of claim 7, wherein the murinized ARHGAP11B gene (Arhgapl laB) encodes the sequenceMWDQRLVRLALLQQLRAVYGIKVKGGRGQCDRRRHETAATEIKGKVFGVPFNS LPHSWPEFGHIPSFLVDACASLKEHIHTEGLFRKSGSVVRLKALKSKLDQGEACL SSALPCDVAGLLKQFFRELPEPVLPADLHEALFKAQQLGAEERNKATLLLSCLMA NPTVDILRYFFNFLKSVSLRASENKMDSSNLAVIFAPNLLQTSEGHEKMSANTEK I<GVYQTLSWI<RYQPCWVLMVSVLLHHWI<ALI<I<VNMI<LLVNIREREDNV (SEQ ID NO: 1).

9. The method of any one of claim 1-5, wherein the murinized human DNA molecule is a murinized NOTCH2NL gene, wherein the murinized NOTCH2NL gene is a chimeric gene named Notch2nlB and encodes the sequence: MPALRPAALRALLWLWLCGAGPAHALQCRGGQEPCVNEGTCVTYHNGTGFCR CPEGFLGEYCQHRDPCEKNRCQNGGTCVPQGMLGKATCRCAPGFTGEDCQYST SHPCFVSRPCQNGGTCHMLSRDTYECTCQVGFTGKQCQWTDACLSHPCENGSTC TSVASQFSCKCPAGLTGQKCEADINECDIPGRCQHGGICLNLPGSYRCQCPQGFT GQHCDSPYVPCAPSPCVNGGTCRQTGDFTFECNCLPETVRRGTELWERDREVWN GKEHDEN (SEQ ID NO: 2.Page 43 of 474918-4489-0203 4065472-000907WQPT10. The method of any one of claims 1-9, wherein the second cistron further comprises one or more murine DNA molecule not expressed in a murine brain.

11. The method of claim 10, wherein the murine DNA molecule not expressed in the murine brain is murine Notch2.

12. The method of claim 10, wherein the murine DNA molecule not expressed in the murine brain is murine Notch2 and encodes SEQ ID NO: 3.

13. The method of any one of claims 1-5, wherein the murinized human DNA molecule is murinized TBC1D3, murinized TMEM14B, or murinized NOTCH2NL.

14. The method of any one of claims 1-13, the donor vector is selected from the group consisting of plasmid, linear PCR, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC).

15. The method of any one of claims 1-14, wherein the donor vector comprises at least four polyadenylation signals upstream from the transgene.

16. The method of any one of claims 1-15, wherein the donor vector further comprises a post- transcriptional regulatory element.

17. The method of any one of claims 1-16, wherein the donor vector further comprises a polyadenylation signal downstream from the transgene.

18. The method of any one of claims 1-17, wherein the donor vector further comprises an open reading frame (ORF) that begins with a splice acceptor.

19. The method of any one of claims 1-18, wherein the donor vector further comprises a fluorescent reporter.

20. The method of any one of claims 1-19, wherein the viral vector is an adeno-associated viral (AAV) vector.

21. The method of any one of claims 1-20, wherein the expression vector comprising recombinases are under tissue-specific promoters.

22. The method of any one of claims 1-21, wherein the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp, modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp,VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, orSloxP and flippase recognition target (FRT), and the recombinases are SCre and flp.

23. The method of any one of claims 1-21, wherein the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively.Page 44 of 474918-4489-0203 4065472-000907WOPT24. The method of any one of claims 1-23, wherein one or both of the paired recombinase recognition sites comprise a mutation.

25. The method of any one of claims 1-24, wherein the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, or Hprt locus.

26. The method of any one of claims 1-25, wherein the paired recombinase recognition sites comprises at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

27. The method of claim 26, wherein the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site.

28. The method of claim 26, wherein the unidirectional recombinase recognition site is downstream to a promoter.

29. The method of claim 26, wherein the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

30. The method of claim 26, wherein the unidirectional recombinase is selected from Bxbl, Phic31, PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, WP, BL3, SPBc, K38, and any mutants thereof.

31. The method of claim 26, wherein the bidirectional recombinase is Flp.

32. The method of claim 26, wherein the unidirectional recombinase is Bxbl and the bidirectional recombinase is selected from Flp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof.

33. The method of claim 26, wherein the unidirectional recombinase recognition site is attB or attP.

34. The method of claim 26, wherein the bidirectional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

35. The method of claim 26, wherein one or both of the recombinase recognition sites comprise a mutation.

36. The method of claim 26, wherein the donor vector comprises an intron, or part of an intron upstream and / or downstream from the transgene, wherein the transgene encodes an RNA.

37. The method of claim 36, wherein the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA.

38. A non -human transgenic rodent, made by any one of the methods of claims 1-37.

39. The transgenic rodent of claim 38, wherein the transgenic rodent further carries a humanspecific gene in its whole genome.Page 45 of 474918-4489-0203 4065472-000907WQPT40. A method of assessing the effects of a drug candidate, comprising: providing the non-human rodent model of claim 38 or claim 39; administering the drug candidate to the non-human rodent model; and assessing the effects of the drug candidate on the non-human rodent model.Page 46 of 474918-4489-0203 4065472-000907WQPT

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