A method for constructing a mammalian target of rapamycin autologous mouse model and mouse embryonic fibroblast cell line and application thereof

Abstract

The application belongs to the technical field of cell engineering, and particularly relates to a mammalian target of rapamycin (mTOR) self-activation mouse model and a construction method and application of a mouse embryonic fibroblast cell line. In the application, a serine (S) at position 2215 of an mTOR protein is mutated into a tyrosine (Y), and a transgenic mouse model of an mTORS2215Y activation mutation, i.e. a mammalian target of rapamycin (mTOR) self-activation mouse model, is constructed. The transgenic mouse model is used to construct a mouse embryonic fibroblast cell line which can be immortalized, so that the related mechanism research of the mTOR self-activation mutation can be carried out at a cell level, and the screening of related therapeutic drugs can be carried out.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell engineering, and particularly relates to a mammalian target of rapamycin (mTOR) self-activation mouse model and a construction method and application of a mouse embryonic fibroblast cell line. BACKGROUND

[0002] Mammalian target of rapamycin (mTOR) is a typical serine / threonine protein kinase, belonging to the PI3K-related kinase (PI3K-related kinase, PIKK) superfamily. mTOR is a main regulatory protein of metabolism and a central node of cell growth network, and plays an important role in maintaining cell ecology and embryonic development. Abnormal mTOR signaling pathway causes diseases such as tumor, metabolic disease, nervous system disease, genetic disease, autoimmune disease and aging. Since knocking out mTOR leads to cell and embryo death, in the research of mTOR, the upstream signaling pathway is activated or inhibited, and mTOR inhibitors are used to indirectly infer the function of mTOR. However, the research on the self-activation of mTOR gene is relatively less, and the exact physiological function and pathological mechanism of mTOR are still unclear. Therefore, it is particularly important to construct a cell line for studying the self-activation of mammalian target of rapamycin. SUMMARY

[0003] The present application aims to provide a construction method and application of a mammalian target of rapamycin (mTOR) self-activation mouse model and a mouse embryonic fibroblast cell line. The mouse model is used to construct a mouse embryonic fibroblast cell line that can be immortalized, so that the related mechanism of mTOR self-activation mutation can be studied at the cell level, and the related therapeutic drugs can be screened.

[0004] The present application provides a construction method of a mammalian target of rapamycin (mTOR) self-activation mouse model, comprising the following steps: connecting an insertion sequence to a homologous arm, and then constructing the insertion sequence into a homologous recombination vector to obtain a homologous recombination targeting vector; transferring the homologous recombination targeting vector into ES cells to construct a hybrid mTORS2215Y mutant mouse mTORS2215Y f / + ; crossing the hybrid mTORS2215Y mutant mouse mTORS2215Y f / + with a wild-type mouse, and backcrossing to screen a homozygous mTORS2215Y mutant mouse mTORS2215Y f / f , to obtain the mammalian target of rapamycin (mTOR) self-activation mouse model.

[0005] The nucleotide sequence of the insertion sequence is shown as SEQ ID NO: 1.

[0006] As a preferred solution, the homologous arm comprises a 5' homologous arm and a 3' homologous arm; the sequence of the 5' homologous arm is the sequence of 90509-95508 bp of the mTOR genomic sequence; and the sequence of the 3' homologous arm is the sequence of 95974-100973 bp of the mTOR genomic sequence.

[0007] As a preferred solution, the mTOR genomic sequence is located on mouse chromosome 4 NC_000070.7, and the Gene ID number of the mTOR gene is 56717.

[0008] The application further provides application of the mammalian mTOR self-activation mouse model obtained by the construction method in preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signaling pathway.

[0009] The application further provides a construction method of a mammalian mTOR self-activation mouse embryonic fibroblast cell line, comprising the following steps: backcrossing the mammalian mTOR self-activation mouse model obtained by the construction method, extracting fibroblasts to obtain fibroblast mTORS2215Y f / f Mefs; the fibroblast mTORS2215Y f / f Mefs is passed to the P4 generation and mixed with a slow virus containing SV40largeT to obtain immortalized fibroblast mTORS2215Y f / f Mefs; the P6 generation of the immortalized fibroblast mTORS2215Y f / f Mefs is mixed with a cre adenovirus to obtain the mammalian mTOR self-activation mouse embryonic fibroblast cell line.

[0010] As a preferred solution, the fibroblasts are taken from mouse embryos, and the mouse embryos are mouse embryos of 13.5 days after backcrossing the mammalian mTOR self-activation mouse model.

[0011] As a preferred solution, the multiplicity of infection MOI of the slow virus containing SV40largeT is 2, and the mixing time of the slow virus containing SV40largeT is 48 h.

[0012] As a preferred solution, the dosage of the cre adenovirus is 1×10 10 pfu / mL, and the mixing time of the cre adenovirus is 48 h.

[0013] The application also provides a mammal mTOR self-activation mouse embryonic fibroblast cell line obtained by the construction method.

[0014] The application also provides application of the mammal mTOR self-activation mouse embryonic fibroblast cell line obtained by the construction method or the mammal mTOR self-activation mouse embryonic fibroblast cell line in preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by mTOR signaling pathways.

[0015] Beneficial effects: the application provides a construction method of a mammal mTOR self-activation mouse model, which comprises the following steps: after connecting an insertion sequence to homologous arms, the insertion sequence is constructed into a homologous recombination vector to obtain a homologous recombination targeting vector; the homologous recombination targeting vector is transferred into ES cells to obtain a hybrid mTORS2215Y mutant mouse mTORS2215Y f / + ; the hybrid mTORS2215Y mutant mouse mTORS2215Y f / + is crossed with a wild-type mouse, and after backcrossing, a homozygous mTORS2215Y mutant mouse mTORS2215Y f / f is screened to obtain the mammal mTOR self-activation mouse model; the nucleotide sequence of the insertion sequence is shown in SEQ ID NO: 1. In the application, Serine (S) at position 2215 of the mTOR protein is mutated into Tyrosine (Y), and a transgenic mouse model of mTORS2215Y activation mutation is constructed. The transgenic mouse model is used to construct a mouse embryonic fibroblast cell line that can be immortalized, so that the related mechanism of mTOR self-activation mutation can be researched at the cell level, and the related therapeutic drugs can be screened. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0017] Figure 1 Construction of the targeting vector in the embodiments;

[0018] Figure 2 Construction of the homologous sequence map of the targeting vector in the embodiments;

[0019] Figure 3 Identification results of the mTOR fLoxP / + mouse in the embodiments;

[0020] Figure 4Blast results of amplified sequences in examples;

[0021] Figure 5 Identification results of mTORmut cells in examples;

[0022] Figure 6 Detection results of mTOR signaling pathway related proteins of mTORmut cells in experimental examples;

[0023] Figure 7 Detection results of proliferation of mTORmut cells in experimental examples;

[0024] Figure 8 Results of plate cloning experiments of mTORmut cells in experimental examples, wherein A is a plate map and B is a cell colony formation number statistical chart;

[0025] Figure 9 Results of nude mouse tumorigenicity detection of mTORmut cells in experimental examples, wherein A is a tumor-free rate statistical result and B is a survival rate statistical result;

[0026] Figure 10 Results of Western blot experiments showing that mTORmut cells are sensitive to mTOR inhibitor rapamycin in experimental examples;

[0027] Figure 11 Effects of rapamycin on the inhibition rate of mTORmut cell tumorigenesis in experimental examples, wherein A is a phenotype chart, B is a tumor weight statistical result, and C is a tumor fold change statistical result;

[0028] Figure 12 Detection results of p-AKT protein in the mTOR signaling pathway of mTORmut cells in experimental examples, wherein A is that TSC2 deletion leads to down-regulation of p-AKT protein, and B is that mTORmut cells up-regulate p-AKT protein expression;

[0029] Notes: Figures 5-12 mTORmut cells in the examples refer to mTORS2215Y mutant cells (mTORmut) and also refer to a mammalian target of rapamycin autophosphorylation mouse embryonic fibroblast cell line. DETAILED DESCRIPTION

[0030] The present application provides a construction method of a mammalian target of rapamycin autophosphorylation mouse model, comprising the following steps: after inserting a sequence into a homologous arm, the sequence is connected to a homologous recombination vector to obtain a homologous recombination targeting vector; the homologous recombination targeting vector is transferred into an ES cell to obtain a hybrid mTORS2215Y mutant mouse mTORS2215Y f / +hybridized mTORS2215Y mice mTORS2215Y f / + After crossing with wild type mice, backcrossing, and screening, homozygous mTORS2215Y mutant mice mTORS2215Y f / f , to obtain the mammalian mTOR autophosphorylation mouse model; the nucleotide sequence of the inserted sequence is shown in SEQ ID NO: 1.

[0031] As a specific embodiment, the homologous arms include a 5' homologous arm and a 3' homologous arm; the sequence of the 5' homologous arm is the sequence of 90509-95508 bp of the mTOR genomic sequence; the sequence of the 3' homologous arm is the sequence of 95974-100973 bp of the mTOR genomic sequence. As a specific embodiment, the mTOR genomic sequence is located on mouse chromosome 4 NC_000070.7, and the Gene ID number of the mTOR gene is 56717.

[0032] As a specific embodiment, the nucleotide sequence containing part of the 5' homologous arm sequence + the inserted sequence + part of the 3' homologous arm sequence is shown in SEQ ID NO: 2. Among them, 1-187 bp is part of the 5' homologous arm, and 5877-5980 bp is part of the 3' homologous arm. 188-221 bp and 5355-5388 bp are LoxP sites, 3509-3542 bp and 5303-5336 bp are Frt sites. 558-1681 bp is the inserted cDNA region of exon 47-58, 1682-2504 bp is the 3'UTR region, 2770-3469 bp is the 3X stop code to terminate the expression after the cDNA region of exon 47-58, so as to express a normal mTOR gene. 3543-5302 bp is a neomycin resistance screening tag (neo cassette), only ES cells screened by neomycin are cells that have undergone homologous recombination. 5741-5876 bp is the 47th exon after gene mutation (5858 bp bases are mutated from C to A).

[0033] The application also provides the use of the mammalian target of rapamycin (mTOR) self-activating mouse model obtained by the construction method in the preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signaling pathway. It should be noted that the drugs of the application are drugs for treating various diseases caused by the mTOR signaling pathway, and any drug for treating diseases caused by the mTOR signaling pathway is a drug of the application. It should be understood that the drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signaling pathway are only several specific embodiments that can achieve the effects of the application, but are not limited to these drugs, and are not specifically limited herein.

[0034] The application also provides a construction method of a mammalian target of rapamycin (mTOR) self-activating mouse embryonic fibroblast cell line, comprising the following steps: backcrossing the mammalian target of rapamycin (mTOR) self-activating mouse model obtained by the construction method, extracting fibroblasts to obtain fibroblast mTORS2215Y f / f Mefs; the fibroblast mTORS2215Y f / f Mefs is passed to the P4 generation and mixed with a slow virus containing SV40largeT to obtain immortalized fibroblast mTORS2215Y f / f Mefs; the P6 generation of immortalized fibroblast mTORS2215Y f / f Mefs is mixed with cre adenovirus to obtain the mammalian target of rapamycin (mTOR) self-activating mouse embryonic fibroblast cell line.

[0035] As a specific embodiment, the fibroblasts are taken from mouse embryos, and the mouse embryos are mouse embryos of 13.5 days after backcrossing the mammalian target of rapamycin (mTOR) self-activating mouse model. As a specific embodiment, the multiplicity of infection (MOI) of the slow virus containing SV40largeT is 2, and the mixing time of the slow virus containing SV40largeT is 48 h. As a specific embodiment, the amount of the cre adenovirus is 1×10 10 pfu / mL, and the mixing time of the cre adenovirus is 48 h.

[0036] The mating scheme of the mouse of the present application involves two recombination systems, Flp-FRT system and Cre-LoxP system. In the Flp-FRT system, Flp (flippase recombination enzyme) recombination enzyme can specifically recognize FRT sequence [5'-gaagttcctattctctagaaagtataggaacttc-3', (SEQ ID NO: 3)], which is composed of two 13 bp inverted palindromic sequences and an 8 bp spacer sequence. The two inverted palindromic sequences adjacent to the spacer sequence are the recognition and binding regions of the Flp recombination enzyme, and the spacer sequence is the region where recombination occurs, which also determines the direction of the entire sequence. In the design of the mouse of the present application, the directions of the FRT sequences are consistent, so that the cutting occurs, thereby cutting off the sequence (neomycin resistance selection tag) in the two FRT sites, leaving only one complete FRT sequence in the genome, thereby removing the neomycin resistance selection tag. In the Cre-LoxP system, Cre recombination enzyme can specifically recognize LoxP sequence [5'-ataacttcgtataatgtatgctatacgaagttat-3', (SEQ ID NO: 4)], which is a 34 bp sequence in P1 phage and is composed of two 13 bp inverted palindromic sequences and an 8 bp asymmetric spacer sequence. The inverted palindromic sequences are the recognition and binding regions of the Cre recombination enzyme, and the spacer sequence is asymmetric, which defines the direction of the LoxP sequence. Cutting and exchange occur within the spacer sequence. In the design of the mouse of the present application, the directions of the LoxP sequences are consistent, so that the cutting occurs, thereby cutting off the sequence (exon 47-58 cDNA sequence, 3' UTR sequence and 3X stop region sequence) in the two LoxP sites, leaving only one complete LoxP sequence in the genome, thereby allowing the 47th exon after the LoxP site which has been mutated from C to A and the 48th-58th exons to be expressed.

[0037] The present application also provides a mammalian mTOR autologous mouse embryonic fibroblast cell line obtained by the construction method.

[0038] The application also provides the use of the mammalian rapamycin target protein self-activating mouse embryonic fibroblast cell line obtained by the construction method in the preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signal pathway. It should be noted that the drug of the application is a drug for treating various diseases caused by the mTOR signal pathway. It can be understood that any drug for treating diseases caused by the mTOR signal pathway is the drug of the application. It can be understood that the drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signal pathway are only several specific embodiments that can achieve the effect of the application, but are not limited to these drugs, and are not specifically limited here.

[0039] The experiments of the application show that the mTOR activation mutation up-regulates the expression of mTOR signal pathway related proteins p-mTOR 2448 , p-mTOR 2481 and p-p70s6k downstream of mTOR; the mTOR activation mutation significantly promotes the proliferation of cells; the mTOR activation mutation promotes the formation of cell plate clones; the mTOR activation cell promotes the tumorigenesis of nude mice and reduces the survival period of mice; the mTORmut cell is sensitive to the mTOR inhibitor rapamycin, and the tumor inhibition rate reaches 59.03%.

[0040] In order to further illustrate the application, the construction method and application of the mammalian rapamycin target protein self-activating mouse model and mouse embryonic fibroblast cell line provided by the application are described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0041] Unless otherwise specified, the raw materials for preparing the application have no special requirements, and commercially available goods known to those skilled in the art can be used.

[0042] 1. The primer sequence used in the application is shown in Table 1.

[0043] Table 1 Primer sequence

[0044]

[0045] LC-mTOR-A1LoxP-F / LC-mTOR-A2LoxP-R: This pair of primers is used to confirm whether the LoxP site has been recombined into the genome, two primers are designed on both sides of a LoxP site; for hybrid animals, two products will be obtained when PCR is performed using this pair of primers: PCR product of wild-type allele, PCR product of mutant allele. Therefore, using this pair of primers can distinguish the genotype of the animal: homozygote / heterozygote / wild type.

[0046] LC-mTOR-Frt-F / LC-mTOR-Frt-R This pair of primers is used to confirm whether the neomycin resistance tag has been removed. When PCR is performed using this pair of primers, the PCR product of the mutant allele can be obtained.

[0047] Flp-F / Flp-R This pair of primers is used to confirm whether the Flp gene exists, and the genotype of Flp positive mice is uniformly represented by Flp / +.

[0048] LC-mTOR-Mut-F / LC-mTOR-Mut-R This pair of primers is used for PCR sequencing to confirm that a C to A mutation has indeed occurred in exon 47.

[0049] LC-mTOR-A1LoxP-F / LC-mTOR-Mut-R This pair of primers is used to better distinguish mTORf / f (no band) and mTORmut (708 bases) cell lines.

[0050] 2. The PCR identification used in the application is mouse tail lysis solution Direct PCR Lysis Reagent (mouse tail) (American Viagen Biotech Company, item number 102-T), proteinase K (Genview, EP264-100MG), 2x Taq PCR StarMix (Beijing Kangrunchengye Biotechnology Co., Ltd., item number A012-10).

[0051] Usage of mouse tail lysis solution: add 100 μL of mouse tail lysis solution and 5 μL of proteinase K to each mouse tail, and place it in a 55°C water bath overnight. The next day, denature at 85°C for 40 min.

[0052] The PCR reaction system (20 µL) consisted of: 10 µL of 2×Taq PCR StarMix, 1 µL of 10 μM Primer-F, 1 µL of 10 μM Primer-R, 6 µL of mQH2O, and 2 µL of DNA. The PCR program was as follows: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 sec, 57 °C annealing for 30 sec, 72 °C extension for 30 sec, for 35 cycles; 72 °C final extension for 10 min, followed by 4 °C for 10 min. After PCR, bands were separated by 3% agarose gel electrophoresis.

[0053] 3. The mTORmut cells of this invention refer to cells with the mTORS22115Y mutation (mTORmut), and also to mouse embryonic fibroblast cell lines that are activated by the mammalian target of rapamycin.

[0054] Example

[0055] 1. Mouse mTOR (mechanistic target of rapamycin kinase [ Mus musculus The (housemouse) gene is located on chromosome 4, NC_000070.7 (148533039..148642142), with a Gene ID of 56717. The gene is 109,104 bases long (https: / / www.ncbi.nlm.nih.gov / gene / 56717), contains 58 exons, and has a coding region of 7,650 base pairs (bp) and 2,549 amino acids (aa). The protein has a molecular weight of approximately 289 kilodaltons (kDa) (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_020009.2). A mutation was made at amino acid position 2215 of mTOR (located in exon 47) to tyrosine (S2215Y, TCT to TAT) to construct a transgenic mouse model of mTORS2215Y activation mutation (i.e., a mouse model of mammalian target of rapamycin self-activation).

[0056] Biocytogen (https: / / www.biocytogen.com.cn / about-us / profile.html) was commissioned to construct a homologous recombination targeting vector for mTOR (S2215Y, TCT to TAT) (e.g., Figure 1 As shown), the mTORS2215Y mutant mouse was successfully constructed by electroporation into ES cells with a c57bl / 6N background.f / + ).

[0057] Construction of homologous sequences inserted into the targeting vector, such as Figure 2 As shown, the targeting sequence mainly includes a 5' homologous arm sequence and a 3' homologous arm sequence, two LoxP sites, an insertion fragment containing normal exons 47-58 cDNA, a 3X STOP region to terminate further expression of this fragment, a neomycin (neo) resistance selection tag, and two Frt sites. The 5' homologous arm sequence, located at bases 90509-95508 of the mTOR genome sequence, contains exons 45 and 46 of the mTOR gene and the primer sequence LC-mTOR-A1LoxP-F for mouse genotyping. The 3' homologous arm sequence, located at bases 95974-100973 of the mTOR genome sequence, contains exons 48-51 of the mTOR gene and the primer sequence LC-mTOR-Mut-R for mouse genotyping. The 5689-base sequence between the two homologous arms is a homologous recombination sequence. The nucleotide sequence containing a partial 5' homologous arm sequence + an insertion sequence + a partial 3' homologous arm sequence is shown in SEQ ID NO:2.

[0058] The F0 generation mice (containing two FRT and LoxP sites) provided by Biocytogen have been mated with Flp-deleter mice, and the neomycin resistance tag sequence between the two FRT sites has been deleted, leaving only one FRT site. These mice are mTOR mice. fLoxP / + Flp / + mice, therefore the identification results for this mouse are as follows: Figure 3 As shown. The identification results include mTOR. fLoxP The sequences and WT sequences, FRT sites, and Flp recombinase sequences were analyzed. The neomycin resistance tag needed to be removed to produce the Frt band (193 bp); otherwise, the sequence length containing the neomycin resistance tag between the Frt primers was 1987 bases, while 2×Taq PCRStarMix amplification only amplified 1000 bases in 30 seconds, resulting in no band. Identification results showed that all four mice amplified the wild-type 234 bp and the mutant 309 bp sequences, indicating that all four mice were heterozygous.

[0059] The PCR products obtained using the LC-mTOR-Mut-F / LC-mTOR-Mut-R primer pair were sent to Qingke Biotechnology for sequencing using LC-mTOR-Mut-F as the sequencing primer. The sequencing results were then subjected to BLAST. The BLAST results are shown below. Figure 4, the results show that No. 1 is normal exon 47, No. 2-5 are the results of the four mice sent for sequencing, and No. 6 is the sequence predicted to have a C to A mutation in exon 47. It can be seen that the four mice do have a C to A mutation.

[0060] 2. The mTORS2215Y mutant mice (mTOR fLoxP / + ; Flp / +) obtained are expanded with WT mice, and the mTORS2215Y fLoxP / + (mTORS2215Y f / + ) mice obtained are backcrossed to obtain homozygous mTORS2215Y f / f mice. The homozygous mTORS2215Y f / f homozygous mice are backcrossed, and 13.5 days after the plug is detected, the mouse embryos are taken to obtain mTORS2215Y f / f embryonic fibroblasts (mTORS2215Y f / f Mefs) (referred to as P0 generation). When passed to P4 generation, SV40 large T-containing lentivirus (pGMLV-SV40T Lentivirus, purchased from Genomed Biotech (Shanghai) Co., Ltd., item number GM-0220LV10-1) is added to the cell culture medium (MOI = 2) to infect for 48 h, and then the DMEM complete medium is replaced. In the mTORS2215Y f / f Mefs of P6 generation, cre adenovirus (AdV5-CMV-Cre-mCMV-copGFP) (1 x 10 10 pfu / mL) and control virus (AdV5-CMV-mCMV-copGFP) (purchased from Weizhen Biotechnology, item number AD201001) are added to act for 48 h, the DMEM complete medium is replaced, and the green fluorescence expression is observed under a microscope to be about 90%. Then, single cell clones are plated in 96-well plates, and the proliferating single clones are selected for pcr identification. The identification results are shown in Figure 5 Lane 1 is the marker of L2000 (Takara, item number 3427A); lane 2 is the negative control; lane 3 is the control of the wild-type mouse; lane 4 is the control of the mTOR f / + mouse; lanes 5, 6, and 7 are mTOR f / f cells (2F3, 3B4, and 5B5 are cell clone numbers); and lanes 8, 9, and 10 are single cell clones (3G3, 7A11, and 3B3 are cell clone numbers) that have the mTORS2215Y mutation. The results confirm that the mTORS22115Y mutant cells (mTORmut) (i.e., the mammalian target of rapamycin autophosphorylation mouse embryonic fibroblast cell line) and the control cells (mTORf / f ).

[0061] Experimental Example

[0062] 1. Western blot was performed on the mTOR S2211 5Y mutant cells (mTORmut) obtained in the example (Zhang H, Chen H, Wang Y. Effects of PKM2 gene silencing on the biological characteristics of human lung cancer cells [J]. Journal of Medical Research, 2018, 47(9): 63-67. DOI: 10.11969 / j.issn.1673-548X.2018.09.015.). As shown in Figure 6 , it was found that the mTOR-activated mutant cells up-regulated the expression of mTOR signaling pathway-related proteins p-mTOR 2448 , p-mTOR 2481 and p-p70s6k downstream of mTOR.

[0063] 2. Cell proliferation curve detection was performed on the mTOR S2211 5Y mutant cells (mTORmut) obtained in the example (3G3, 7A11 and 3B3) (Zhang H, Chen H, Wang Y. Effects of PKM2 gene silencing on the biological characteristics of human lung cancer cells [J]. Journal of Medical Research, 2018, 47(9): 63-67. DOI: 10.11969 / j.issn.1673-548X.2018.09.015.). As shown in Figure 7 and Table 2, it was found that the mTOR-activated mutation significantly promoted the proliferation of cells.

[0064] Table 2 Results of cell proliferation curve detection

[0065]

[0066] Taking the proliferation rate of each group of cells at 96h in the above data, one way anova analysis was performed between groups, and the results are shown in Table 3. It can be seen that the proliferation rate of the three cells in the mTORmut group is significantly higher than that of the three cells in the WT group, and the difference between the groups is significant. The proliferation rate of the three cells in the WT group was not significantly different from each other. Although the three cells in the mTORmut group were also different from each other, the proliferation rate of the three cells was higher than that of the cells in the WT group.

[0067] Table 3 Results of one way anova analysis between groups

[0068]

[0069] 3. Take 500 cells from each of the WT group and the mOTRmut group (mOTRmut cells obtained in the examples), respectively, and spread them in 3 10 cm culture dishes, respectively, and culture them for 10 days to perform a plate cloning experiment (Li K, Wang YN. Sodium hydroxide enhances the inhibition of rapamycin on the proliferation of Tsc2 knockout mouse embryonic fibroblasts [J]. Basic Medicine and Clinical, 2021, 41(7):951-956. DOI:10.3969 / j.issn.1001-6325.2021.07.004.). The results are shown in Table 4 and Fig. 2. Figure 8 It is found that the mTOR activation mutation promotes the formation of cell plate clones.

[0070] Table 4. Statistical results of cell clone formation number

[0071]

[0072] 4. Perform a nude mouse tumor formation test (Chen X X, Wang Y N et al., mTORC1 Up-Regulates GP73 to Promote Proliferation and Migration of Hepatocellular Carcinoma Cells and Growth of Xenograft Tumors in Mice, Gastroenterology. 2015 Sep;149(3):741-52.e14.). Take 1x10 6 cells from each of the WT group and the mOTRmut group (mOTRmut cells obtained in the examples), respectively, and implant them subcutaneously in 16 8-week-old nude mice (half male and half female). Take the appearance of a needle tip size tumor in the subcutaneous tissue of the nude mice as the endpoint, record the time when the subcutaneous tumor of the nude mice is free of tumor, and take the subcutaneous tumor volume greater than 1000 mm 3 or necrotic ulcer and body weight reduction of more than 10% as the endpoint, and record the survival period of the mice. The results are shown in Table 4 and Fig. 3. Figure 9 It is found that mTOR-activated cells promote tumor formation in nude mice and reduce the survival period of mice.

[0073] 5. Dissolve rapamycin (purchased from Sigma-Aldrich Company, item number 553210) in methanol, and store it at a concentration of 10 μM, and use it at a working concentration of 10 nM. After treating the cells (wild type cells and mTORmut cells) with 10 nM rapamycin for 24 h, collect the protein samples and perform western blot detection. The results are shown in Table 5 and Fig. 4. Figure 10 It is found that after adding rapamycin for 24 h, the key proteins of the mTOR signaling pathway, p-mTOR 2448 , p-mTOR 2481 , and p-S6 downstream thereof are reduced.235 / 236 Western blot experiments showed that mTORmut cells were sensitive to mTOR inhibitor rapamycin.

[0074] 6. Nude mice tumor formation experiments showed that mTORmut cells were sensitive to mTOR inhibitor rapamycin, and the tumor inhibition rate reached 59.03%. Take 1x10 6 mOTRmut cells, respectively, were implanted subcutaneously in 16 8-week-old nude mice (half male and half female). After the subcutaneous tumors grew to 50-100mm 3 The mice were randomly divided into two groups, one group used adjuvant and the other group used rapamycin at 6mg / kg for intraperitoneal injection, 3 times a week to treat subcutaneous tumors. The endpoint was when the subcutaneous tumor volume of the adjuvant group was greater than 1000mm 3 or had necrotic ulcers and body weight decreased by more than 10%. The change in tumor volume of the mice was recorded, and the tumors were collected and weighed. The preparation method for injection with rapamycin: the storage solution concentration was 20mg / mL dissolved in absolute ethanol. The present use was prepared according to the concentration of 100μg / 100μL, dissolved in 0.25% PEG and 0.25% Tween-20 adjuvant, and injected at a concentration of 6mg / Kg, and the adjuvant group was injected with the same volume of adjuvant, 3 times a week. The results are shown in Figure 11 and Table 5, it was found that the subcutaneous tumors of nude mice caused by mTOR-activated cells were sensitive to mTOR inhibitor rapamycin, and the subcutaneous tumor weight and volume of the rapamycin injection group were smaller than those of the adjuvant group, and the difference was statistically significant. The inhibition rate of rapamycin on mTORmut group tumor was 59.03%.

[0075] Table 5 Statistical results of tumor inhibition rate

[0076]

[0077] 7. The deletion of the tumor suppressor gene TSC1 / 2 upstream of mTOR can also cause the activation of the mTOR signaling pathway, but the deletion of TSC1 / 2 gene will inhibit the expression of p-AKT protein by up-regulating the expression of mTOR gene, which is caused by the inactivation mutation of TSC1 or TSC2 tumor suppressor gene. Tuberous sclerosis is a cause of benign tumor syndrome (Zhang H, Bajraszewski N, Wu E, et al. PDGFRs are critical for PI3K / Akt activation and negatively regulated by mTOR. J Clin Invest. 2007 Mar;117(3):730-8. doi:10.1172 / JCI28984), but for the mTOR-activated cells, the present application finds that the expression of p-AKT protein is up-regulated, which is different from the mechanism of tuberous sclerosis caused by the deletion of TSC1 / 2, and provides a reliable and stable cell model for the study of mTOR-activated diseases. Figure 12 ).

[0078] Therefore, the present application mutates the serine (Serine, S) at position 2215 of the mTOR protein to tyrosine (Tyrosine, Y) to construct an mTORS2215Y activated mutant transgenic mouse model. And using the transgenic mouse model, a mouse embryonic fibroblast cell line that can be immortalized is constructed, so that the related mechanism of mTOR self-activation mutation can be studied at the cellular level, and the screening of related therapeutic drugs can be carried out.

[0079] Although the above embodiment describes the present application in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which belong to the protection scope of the present application.

Claims

1. A method for constructing a mouse model of mammalian target of rapamycin autophagy, characterized by, The method comprises the following steps: After the insertion sequence is connected with the homologous arms, the homologous recombination carrier is constructed to obtain a homologous recombination targeting carrier. The homologous recombination targeting vector is transferred into ES cells to construct a hybrid mTORS2215Y mutant mouse mTORS2215Y f / + ; mTORS2215Y f / + mTORS2215Y f / f , to obtain the mammalian target of rapamycin autologous mouse model; The nucleotide sequence of the insertion sequence is shown in SEQ ID NO:

1.

2. The construction method of claim 1, wherein, The homologous arms comprise 5' homologous arms and 3' homologous arms; the sequence of the 5' homologous arms is the sequence of 90509-95508 bp of the mTOR genomic sequence; and the sequence of the 3' homologous arms is the sequence of 95974-100973 bp of the mTOR genomic sequence.

3. The construction method of claim 2, wherein, The mTOR genomic sequence is located on the mouse chromosome 4 NC_000070.7, and the Gene ID number of the mTOR gene is 56717.

4. Application of the mammalian target of rapamycin autophosphorylation mouse model obtained by the construction method in any one of claims 1-3 in preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signaling pathway.

5. A method for constructing a mammalian mTOR autologous mouse embryonic fibroblast cell line, characterized by, The method comprises the following steps: The mammalian target of rapamycin autophosphorylation mouse model obtained by the construction method in any one of claims 1-3 is backcrossed, and fibroblasts are extracted to obtain fibroblast mTORS2215Yf / f Mefs. mTORS2215Y fibroblasts f / f Mefs were immortalized by mixing with SV40 large T containing lentivirus after passage to P4 f / f Mefs P6 generation immortalized somatic fibroblast mTORS2215Y f / f Mefs were mixed with cre adenovirus to obtain the mammalian target of rapamycin self-activating mouse embryonic fibroblast cell line.

6. The construction method of claim 5, wherein, The fibroblasts are taken from mouse embryos, and the mouse embryos are mouse embryos of 13.5 days after the mammalian target of rapamycin autophosphorylation mouse model is backcrossed.

7. The construction method of claim 5, wherein, The multiplicity of infection MOI of the SV40 large T-containing lentivirus is 2, and the mixing time of the SV40 large T-containing lentivirus is 48 h.

8. The construction method of claim 5, wherein, The cre adenovirus is used in an amount of 1 x 10 10 pfu / mL, and the mixing time of the cre adenovirus is 48 h.

9. A mammalian target of rapamycin autophosphorylation mouse embryonic fibroblast cell line obtained by the construction method in any one of claims 5-8; and the mouse embryonic fibroblast cell line is an immortalized mouse embryonic fibroblast cell line.

10. Application of the mammalian target of rapamycin autophosphorylation mouse embryonic fibroblast cell line obtained by the construction method in any one of claims 5-8 or the mammalian target of rapamycin autophosphorylation mouse embryonic fibroblast cell line in claim 9 in preparation and / or screening of drugs for treating tumors, metabolic diseases, nervous system diseases, genetic diseases, autoimmune diseases or aging caused by the mTOR signaling pathway.

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