Construction method and application of mammal rapamycin target protein self-activated mouse model and mouse embryo fibroblast line

Abstract

The invention belongs to the technical field of cell engineering, and particularly relates to a construction method and application of a mammal rapamycin target protein self-activated mouse model and a mouse embryo fibroblast line. According to the invention, serine (Serine, S) at site 2215 of mTOR protein is mutated into tyrosine (Tyrosine, Y), and a transgenic mouse model with activation mutation of mTORS2215Y, namely a mouse model with self-activation of mammal rapamycin target protein, is constructed. A mouse embryo fibroblast line which can be immortalized is constructed by utilizing the transgenic mouse model, so that the related mechanism research of mTOR self-activation mutation can be carried out at the cellular level, and the screening of related therapeutic drugs can be carried out.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell engineering, and particularly relates to a method for constructing and applying a mouse model with self-activated mammalian target of rapamycin (mTOR) and a mouse embryonic fibroblast cell line. Background Art

[0002] Mammalian target of rapamycin (mTOR) is a typical serine / threonine protein kinase and belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily. mTOR is a major regulatory protein of metabolism and a central node controlling the cell growth network, and plays an important role in maintaining cell homeostasis and embryonic development. Abnormal mTOR signaling pathways cause diseases such as tumors, metabolic diseases, neurological diseases, genetic diseases, autoimmune diseases, and aging. Since knocking out mTOR leads to cell and embryo death, in the study of mTOR, activating or inhibiting upstream signaling pathways and using mTOR inhibitors are mostly used to indirectly infer the function of mTOR, while the study of the self-activation of the mTOR gene is relatively less, and the exact physiological functions and pathological mechanisms of mTOR are still not very clear. Therefore, it is particularly important to construct a cell line for studying the self-activation of mammalian target of rapamycin. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for constructing and applying a mouse model with self-activated mammalian target of rapamycin and a mouse embryonic fibroblast cell line. By using the mouse model of the present invention, an immortalized mouse embryonic fibroblast cell line is constructed, so that the related mechanisms of mTOR self-activation mutation can be studied at the cell level, and related therapeutic drugs can be screened.

[0004] The present invention provides a method for constructing a mouse model with self-activated mammalian target of rapamycin, comprising the following steps: after connecting the insertion sequence with homologous arms, constructing it onto a homologous recombination vector to obtain a homologous recombination targeting vector; transferring the homologous recombination targeting vector into ES cells to construct a heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + ; crossing and backcrossing the heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + with a wild-type mouse, and screening for a homozygous mTORS2215Y mutant mouse mTORS2215Y f / f to obtain the mouse model with self-activated mammalian target of rapamycin; The nucleotide sequence of the insertion sequence is as shown in SEQ ID NO: 1.

[0005] As a preferred 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.

[0006] As a preferred 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.

[0007] The present invention also provides the application of the mouse model with self-activated mammalian target of rapamycin 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.

[0008] The present invention also provides a method for constructing a mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin, comprising the following steps: backcrossing the mouse model with self-activated mammalian target of rapamycin obtained by the construction method, extracting fibroblasts to obtain fibroblast mTORS2215Y f / f Mefs; passing fibroblast mTORS2215Y f / f Mefs to passage P4 and then mixing with lentivirus containing SV40 large T to obtain immortalized fibroblast mTORS2215Y f / f Mefs; mixing the P6-generation immortalized fibroblast mTORS2215Y f / f Mefs with cre adenovirus to obtain the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin.

[0009] As a preferred embodiment, the fibroblasts are taken from a mouse embryo, and the mouse embryo is a mouse embryo at 13.5 days after plug detection after backcrossing the mouse model with self-activated mammalian target of rapamycin.

[0010] As a preferred embodiment, the multiplicity of infection MOI of the lentivirus containing SV40 large T is 2, and the mixing time of the lentivirus containing SV40 large T is 48 h.

[0011] As a preferred embodiment, the dosage of the cre adenovirus is 1×10 10 pfu / mL, and the mixing time of the cre adenovirus is 48 h.

[0012] The present invention also provides a mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin obtained by using the described construction method.

[0013] The present invention also provides the application of the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin obtained by using the described construction method or the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin 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.

[0014] Beneficial effects: The present invention provides a method for constructing a mouse model with self-activated mammalian target of rapamycin, including the following steps: after connecting the insertion sequence with homologous arms, constructing it onto a homologous recombination vector to obtain a homologous recombination targeting vector; transferring the homologous recombination targeting vector into ES cells to construct a heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + ; hybridizing and backcrossing the heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + with wild-type mice, and screening for homozygous mTORS2215Y mutant mice mTORS2215Y f / f to obtain the mouse model with self-activated mammalian target of rapamycin; the nucleotide sequence of the insertion sequence is as shown in SEQ ID NO:1. The present invention mutates serine (Serine, S) at position 2215 of the mTOR protein to tyrosine (Tyrosine, Y) to construct a transgenic mouse model with activated mTORS2215Y mutation. And by using the transgenic mouse model, an immortalized mouse embryonic fibroblast cell line is constructed, so that the related mechanism of mTOR self-activation mutation can be studied at the cellular level, and related therapeutic drugs can be screened. Brief Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0016] Figure 1 For the construction of the targeting vector in the embodiment; Figure 2 For the construction map of the homologous sequence of the targeting vector in the embodiment; Figure 3 For the identification result of the mTOR fLoxP / + mouse in the embodiment; Figure 4 For the Blast result of the amplified sequence in the embodiment; Figure 5Identification results of mTORmut cells in the examples; Figure 6 Detection results of proteins related to the mTOR signaling pathway in mTORmut cells in the experimental examples; Figure 7 Detection results of the proliferation of mTORmut cells in the experimental examples; Figure 8 Results of the plate cloning experiment of mTORmut cells in the experimental examples, where A is the plate diagram and B is the statistical chart of the number of cell clones formed; Figure 9 Results of the tumorigenicity detection of mTORmut cells in nude mice in the experimental examples, where A is the statistical result of the tumor-free rate and B is the statistical result of the survival rate; Figure 10 Results showing that mTORmut cells are sensitive to the mTOR inhibitor rapamycin in the Western blot experiment in the experimental examples; Figure 11 Effect of rapamycin on the inhibition rate of tumorigenicity of mTORmut cells in the experimental examples, where A is the phenotype diagram, B is the statistical result of the tumor weight, and C is the statistical result of the change in tumor multiples; Figure 12 Detection results of the p-AKT protein in the mTOR signaling pathway of mTORmut cells in the experimental examples, where A shows that the downregulation of the p-AKT protein is caused by the deletion of TSC2, and B shows that the expression of the p-AKT protein is upregulated in mTORmut cells; Note: Figures 5 to 12 The mTORmut cells in [] refer to the cells with the mTORS22115Y mutation (mTORmut), and also refer to the mouse embryonic fibroblast cell line with autoactivated mammalian target of rapamycin. Detailed implementation mode

[0017] The present invention provides a method for constructing a mouse model with autoactivated mammalian target of rapamycin, including the following steps: after connecting the insertion sequence with homologous arms, constructing it onto a homologous recombination vector to obtain a homologous recombination targeting vector; transferring the homologous recombination targeting vector into ES cells to construct a heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + ; crossing and backcrossing the heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + with wild-type mice, and screening for homozygous mTORS2215Y mutant mice mTORS2215Y f / f to obtain the mouse model with autoactivated mammalian target of rapamycin; the nucleotide sequence of the insertion sequence is as shown in SEQ ID NO:1.

[0018] 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.

[0019] As a specific embodiment, the nucleotide sequence containing a partial 5' terminal homologous arm sequence + an inserted sequence + a partial 3' terminal homologous arm sequence is shown in SEQ ID NO:2. Among them, 1-187 bp is a part of the 5' homologous arm, and 5877-5980 bp is a part of the 3' homologous arm. 188-221 bp and 5355-5388 bp are LoxP sites, and 3509-3542 bp and 5303-5336 bp are Frt sites. 558-1681 bp is the cDNA region of exons 47-58 inserted, 1682-2504 bp is the 3'UTR region, and 2770-3469 bp is the 3X stop region (3X stop code) used to terminate the expression after the cDNA region of exons 47-58, so as to express a normal mTOR gene. 3543-5302 bp is the neomycin resistance screening tag (neo cassette), and only ES cells screened by neomycin are cells that have undergone homologous recombination. 5741-5876 bp is exon 47 with a gene mutation (where the base at 5858 bp is mutated from C to A).

[0020] The present invention also provides the application of the mammalian target of rapamycin self-activated mouse model obtained by the construction method described above 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 key to the drug of the present invention is a drug for treating various diseases caused by the mTOR signaling pathway. It can be understood that any drug for treating diseases caused by the mTOR signaling pathway is the drug of the present invention. It can be understood that 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 present invention, but are not limited to these drugs, and are not specifically limited herein.

[0021] The present invention also provides a method for constructing a mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin (mTOR), comprising the following steps: backcrossing a mouse model with self-activated mTOR obtained by using the construction method, extracting fibroblasts to obtain fibroblast mTORS2215Y f / f Mefs; passing fibroblast mTORS2215Y f / f Mefs to passage P4 and then mixing with a lentivirus containing SV40 large T to obtain immortalized fibroblast mTORS2215Y f / f Mefs; mixing the immortalized fibroblast mTORS2215Y f / f Mefs at passage P6 with cre adenovirus to obtain the mouse embryonic fibroblast cell line with self-activated mTOR.

[0022] As a specific embodiment, the fibroblasts are taken from a mouse embryo, and the mouse embryo is a mouse embryo at 13.5 days after plug inspection after backcrossing a mouse model with self-activated mTOR. As a specific embodiment, the multiplicity of infection (MOI) of the lentivirus containing SV40 large T is 2, and the mixing time of the lentivirus containing SV40 large T is 48 h. As a specific embodiment, the dosage of the cre adenovirus is 1×10 10 pfu / mL, and the mixing time of the cre adenovirus is 48 h.

[0023] The mating scheme of the mice in the present invention involves two recombination systems, the Flp-FRT system and the Cre-LoxP system. In the Flp-FRT system, the Flp (flippase recombination enzyme) recombinase can specifically recognize the FRT sequence [5’-gaagttcctattctctagaaagtataggaacttc-3’, (SEQ ID NO:3)]. The FRT sequence is composed of two 13bp inverted palindromic sequences and an 8bp spacer sequence. The two inverted palindromic sequences adjacent to the spacer sequence are the recognition and binding regions of the Flp recombinase, and the spacer sequence is the region where recombination occurs and also determines the direction of the entire sequence. In the design of the mice in the present invention, the directions of the FRT sequences are the same, so cleavage occurs, thereby cutting off the sequence (neomycin resistance screening tag) between the two FRT sites, leaving only one complete FRT sequence in the genome, thus removing the neomycin resistance screening tag. In the Cre-LoxP system, the Cre recombinase can specifically recognize the LoxP sequence [5’-ataacttcgtataatgtatgctatacgaagttat-3’, (SEQ ID NO:4)]. LoxP (Locus of X-over P1) is a 34bp sequence located in phage P1 and is composed of two 13bp inverted palindromic sequences and an 8bp intervening spacer sequence. The inverted palindromic sequences are the recognition and binding regions of the Cre recombinase, and the spacer sequence is asymmetric. It is this asymmetry that defines the direction of the LoxP sequence, and cleavage and exchange occur within the spacer sequence. In the design of the mice in the present invention, the directions of the LoxP sequences are the same, so cleavage occurs, thereby cutting off the sequences (exon 47-58 cDNA sequence, 3’UTR sequence and 3X stop region sequence) between the two LoxP sites, leaving only one complete LoxP sequence in the genome, so that exon 47 which has undergone a C to A mutation and exons 48-58 after it can be expressed.

[0024] The present invention also provides a mammalian rapamycin target protein self-activated mouse embryonic fibroblast cell line obtained by using the described construction method.

[0025] The present invention also provides the use of the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin obtained by the described construction method or the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin 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 key to the drugs of the present invention lies in the drugs for treating various diseases caused by the mTOR signaling pathway. It can be understood that any drug for treating diseases caused by the mTOR signaling pathway is the drug described in the present invention. 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 signaling pathway are only several specific embodiments that can achieve the effects of the present invention, but are not limited to these drugs, and are not specifically defined herein.

[0026] Experiments of the present invention show that cells with mTOR activation mutations up-regulate the expression of proteins related to the mTOR signaling pathway, such as p-mTOR 2448 and p-mTOR 2481 as well as p-p70s6k downstream of mTOR; mTOR activation mutations significantly promote cell proliferation; mTOR activation mutations promote the formation of cell plate clones; mTOR-activated cells promote tumor formation in nude mice and reduce the survival period of mice; mTORmut cells are sensitive to the mTOR inhibitor rapamycin, and the tumor inhibition rate reaches 59.03%.

[0027] To further illustrate the present invention, the following examples will be used to describe in detail the construction method and application of a mouse model with self-activated mammalian target of rapamycin and a mouse embryonic fibroblast cell line provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0028] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0029] 1. The primer sequences used in the present invention are shown in Table 1.

[0030] Table 1 Primer Sequences

[0031] 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. The two primers are designed on both sides of a LoxP site respectively; for heterozygous animals, two products will be obtained when using this pair of primers for PCR: the PCR product of the wild-type allele and the PCR product of the mutant allele. Therefore, this pair of primers can be used to distinguish the genotypes of animals: homozygous / heterozygous / wild-type.

[0032] The pair of primers LC-mTOR-Frt-F / LC-mTOR-Frt-R is used to confirm whether the neomycin resistance tag has been removed. When using this pair of primers for PCR, the PCR product of the mutant allele can be obtained.

[0033] The pair of primers Flp-F / Flp-R is used to confirm the presence of the Flp gene. The genotype of Flp-positive mice is uniformly represented by Flp / +.

[0034] The pair of primers LC-mTOR-Mut-F / LC-mTOR-Mut-R is used to sequence the PCR result to clarify that a C-to-A mutation has indeed occurred in exon 47.

[0035] The pair of primers LC-mTOR-A1LoxP-F / LC-mTOR-Mut-R is used to better distinguish between the mTORf / f (no band) and mTORmut (708 bases) cell lines.

[0036] 2. The mouse tail lysis solution Direct PCR Lysis Reagent (mouse tail) (Viagen Biotech, USA, product number 102-T), proteinase K (Genview, EP264-100MG), and 2×Taq PCR StarMix (Beijing Kangrun Chengye Biotechnology Co., Ltd., product number A012-10) are used for PCR identification in the present invention.

[0037] Usage of the mouse tail lysis solution: Add 100 μL of the 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 it at 85°C for 40 min.

[0038] The PCR reaction system (20 μL) is as follows: 2×Taq PCR StarMix 10 μL, 10 μM Primer-F 1 μL, 10 μM Primer-R 1 μL, mQH2O 6 μL, DNA 2 μL; the PCR reaction program is as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; final extension at 72°C for 10 min, at 4°C for 10 min. After PCR, band separation is performed by 3% agarose gel electrophoresis.

[0039] 3. The mTORmut cells of the present invention refer to cells with the mTORS22115Y mutation (mTORmut), and also refer to a mouse embryonic fibroblast cell line with auto-activated mammalian target of rapamycin protein.

[0040] Examples 1. Mouse mTOR (mechanistic target of rapamycin kinase Mus musculus (housemouse)] gene is located at NC_000070.7 (148533039..148642142) on chromosome 4, the Gene ID number is 56717, the full length of the gene is 109,104 base pairs (https: / / www.ncbi.nlm.nih.gov / gene / 56717), contains 58 exons, the full length of the coding region is 7,650 base pairs (base pair, bp), 2,549 amino acids (amino acid, aa), and the molecular weight of the protein is about 289 kilodaltons (kDa) (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_020009.2). A mutation from serine (Serine, S) to tyrosine (Tyrosine, Y) (S2215Y, TCT to TAT) was made at the 2215th amino acid site of mTOR (located in exon 47) to construct a transgenic mouse model with activated mTORS2215Y mutation (i.e., a mouse model with auto-activated mammalian target of rapamycin protein).

[0041] Entrusted with Bioocytogen Co., Ltd. (https: / / www.biocytogen.com.cn / about-us / profile.html), a homologous recombination targeting vector of mTOR (S2215Y, TCT to TAT) was constructed (as Figure 1 shown), electrotransfected into ES cells with a c57bl / 6N background, and a mouse with the mTORS2215Y mutation (mTORS2215Y f / + ) was successfully constructed.

[0042] Construction of the homologous sequences inserted into the targeting vector. As Figure 2 shown, the targeting sequence mainly includes a 5'-terminal homologous arm sequence and a 3'-terminal homologous arm sequence, two LoxP sites, an inserted fragment containing the cDNA of exons 47-58 in the normal form, a 3X STOP region that terminates the continuous expression of this fragment, a resistance screening tag for neomycin (neo), and two Frt sites. Among them, the 5'-terminal homologous arm sequence is located at the 90509-95508th base of the mTOR genomic sequence, contains exons 45 and 46 of the mTOR gene, and also contains the primer sequence LC-mTOR-A1LoxP-F for mouse genotype identification. The 3'-terminal homologous arm sequence is located at the 95974-100973th base of the mTOR genomic sequence, contains exons 48-51 of the mTOR gene, and the primer sequence LC-mTOR-Mut-R for mouse genotype identification. The sequence of 5689 bases between the two homologous arms is the homologous recombination sequence. The nucleotide sequence containing part of the 5'-terminal homologous arm sequence + inserted sequence + part of the 3'-terminal homologous arm sequence is shown in SEQ ID NO:2.

[0043] The F0 generation provided by Beijing Biocytogen (containing two Frt and LoxP sites) has been mated with Flp-deleter mice to delete the sequence of the neomycin resistance tag between the two Frt sites, and only the mouse with one remaining Frt site is mTOR fLoxP / + ; Flp / + mouse. Therefore, the identification results of this mouse are as Figure 3 shown. The identification results include the mTOR fLoxP sequence and the WT sequence, the Frt site and the Flp recombinase sequence. The neomycin resistance tag needs to be deleted to see the Frt band (193bp). Otherwise, the sequence length between the Frt primers containing the neomycin resistance tag is 1987 bases, and the 2×Taq PCRStarMix can only amplify 1000 bases in 30s and cannot obtain a band. The identification results show that wild-type 234bp and mutant 309bp were amplified from all 4 mice, indicating that all 4 mice are heterozygous mice.

[0044] Using the pair of primers LC-mTOR-Mut-F / LC-mTOR-Mut-R, the obtained PCR product was sent to Tsingke Biological for sequencing with LC-mTOR-Mut-F as the sequencing primer. The sequencing results were subjected to Blast. The Blast results are shown in Figure 4, The results showed that No. 1 was the normal exon 47, Nos. 2 - 5 were the results of 4 mice sent for sequencing, and No. 6 was the sequence predicted to have a C to A mutation in exon 47. It can be seen that there is indeed a C to A mutation in the 4 mice.

[0045] 2. Using the obtained mice with the mTORS2215Y mutation (mTOR fLoxP / + ; Flp / +), after expanding with WT mice, the obtained mTORS2215Y fLoxP / + (mTORS2215Y f / + ) mice were backcrossed to obtain homozygous mTORS2215Y f / f mice. Using the homozygous mTORS2215Y f / f homozygous mice for backcrossing, at 13.5 days after plug checking, mouse embryos were taken to extract mTORS2215Y f / f mouse embryonic fibroblasts (mTORS2215Y f / f Mefs) (recorded as the P0 generation). When passed to the P4 generation, lentivirus containing SV40 large T (pGMLV - SV40T Lentivirus purchased from GeneCopoeia Biotechnology (Shanghai) Co., Ltd., product number GM - 0220LV10 - 1) (MOI = 2) was added to the cell culture medium and infected for 48 h, then replaced with DMEM complete medium. In the mTORS2215Y f / f Mefs at the P6 generation, cre adenovirus (AdV5 - CMV - Cre - mCMV - copGFP) (1×10 10 pfu / mL) and control virus (AdV5 - CMV - mCMV - copGFP) (purchased from Vigene Biosciences, product number AD201001) were added, and the cells were treated for 48 h, then replaced with DMEM complete medium, and the green fluorescence expression was observed under the microscope to be about 90%. Then, single - cell clones were plated in 96 - well plates, and single - cell clones that could proliferate were selected for pcr identification. The identification results are shown in Figure 5 , Lane 1 is the L2000 marker (Takara, product number 3427A); Lane 2 is the negative control; Lane 3 is the control of wild - type mice; Lane 4 is the control of mTOR f / + mice; Lanes 5, 6, 7 are mTOR f / f cells (2F3, 3B4, 5B5 are cell clone numbers); Lanes 8, 9, 10 are single - cell clones with the mTORS2215Y mutation (3G3, 7A11, 3B3 are cell clone numbers). The results confirmed the obtained cells with the mTORS22115Y mutation (mTORmut) (i.e., the mouse embryonic fibroblast cell line with self - activation of mammalian target of rapamycin), and control cells (mTORf / f ).

[0046] Experimental Example 1. The cells with mTOR S22115Y mutation (mTORmut) obtained in the examples were subjected to western blot (Zhang Haihong, Chen Hongyu, Wang Yanan. Effects of PKM2 gene silencing on 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.). The results are as Figure 6 shown, and it was found that the cells with mTOR activation mutation up-regulated the expression of proteins related to the mTOR signaling pathway, p-mTOR 2448 and p-mTOR 2481 as well as p-p70s6k downstream of mTOR.

[0047] 2. The cells with mTOR S22115Y mutation (mTORmut) (3G3, 7A11, and 3B3) obtained in the examples were subjected to detection of cell proliferation curves (Zhang Haihong, Chen Hongyu, Wang Yanan. Effects of PKM2 gene silencing on 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.). The results are as Figure 7 and Table 2 shown, and it was found that mTOR activation mutation significantly promoted cell proliferation.

[0048] Table 2 Detection results of cell proliferation curves

[0049] Among the above data, the proliferation multiples of each group of cells at 96 h were analyzed by one-way ANOVA between groups. The results are shown in Table 3. It can be seen that the proliferation rates of the 3 cells in the mTORmut group were significantly higher than those of the 3 cells in the WT group, and the difference between groups was significant. There was no obvious difference in pairwise comparison of the proliferation rates of the 3 cells in the WT group. Although there were also differences in pairwise comparison of the 3 cells in the mTORmut group, the proliferation rates of the 3 cells were all higher than those of the cells in the WT group.

[0050] Table 3 Analysis results of one-way ANOVA between groups

[0051] 3. Take 500 cells each from the WT group and the mOTRmut group (mOTRmut cells obtained in the examples), and seed each group into 3 10-cm culture dishes respectively. Culture for 10 days and perform the colony formation assay (Li Kai, Wang Yanan. Sodium hydroxide enhances the inhibitory effect of rapamycin on the proliferation of mouse embryonic fibroblasts lacking Tsc2 [J]. Basic & Clinical Medicine, 2021, 41(7): 951-956. DOI: 10.3969 / j.issn.1001-6325.2021.07.004.). The results are as Figure 8 shown in Table 4, and it was found that the mTOR activation mutation promoted the formation of cell colonies.

[0052] Table 4 Statistical results of the number of cell colonies formed

[0053] 4. Perform nude mouse tumorigenicity detection (Chen Xinxin, Wang Yanan, 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 1×10 6 cells each from the WT group and the mOTRmut group (mOTRmut cells obtained in the examples), and implant them subcutaneously into 16 8-week-old nude mice (8 males and 8 females). Using the appearance of a pinhead-sized tumor under the skin of the nude mouse as the endpoint, record the time when there is no tumor under the skin of the nude mouse. Using the subcutaneous tumor volume greater than 1000 mm 3 or the presence of necrosis, ulceration, and a weight loss of more than 10% as the endpoint, record the survival period of the mice. The results are as Figure 9 shown, and it was found that mTOR-activated cells promoted tumorigenesis in nude mice and shortened the survival period of the mice.

[0054] 5. Dissolve rapamycin (purchased from Sigma-Aldrich, catalog number 553210) in methanol. The storage concentration is 10 μM, and the working concentration is 10 nM. After treating cells (wild-type cells and mTORmut cells) with 10 nM rapamycin for 24 h, collect protein samples and perform western blot detection. The results are as Figure 10 shown, and it was found that after adding rapamycin for 24 h, the key proteins p-mTOR 2448 and p-mTOR 2481 in the mTOR signaling pathway, as well as its downstream p-S6 235 / 236Significantly reduced. Western blot experiments showed that mTORmut cells were sensitive to the mTOR inhibitor rapamycin.

[0055] 6. The xenograft tumor experiment in nude mice showed that mTORmut cells were sensitive to the mTOR inhibitor rapamycin, and the tumor inhibition rate reached 59.03%. Take 1×10 6 mOTRmut cells and implant them subcutaneously into 16 8-week-old nude mice (half male and half female). When the subcutaneous tumor grew to 50 - 100 mm 3 , the mice were randomly divided into two groups. One group was treated with adjuvant, and the other group was injected intraperitoneally with rapamycin at a dose of 6 mg / kg three times a week to treat the subcutaneous tumor. The endpoint was defined as the subcutaneous tumor volume in the adjuvant group being greater than 1000 mm 3 or having necrosis, ulceration, and a weight loss of more than 10%. Record the change in the tumor volume of the mice and collect the weighed tumors of the mice. Preparation method of injectable rapamycin: Stock solution concentration: 20 mg / mL dissolved in absolute ethanol. Prepare it freshly according to a concentration of 100 μg / 100 μL, dissolve it in an adjuvant containing 0.25% PEG and 0.25% Tween-20, and inject it at a concentration of 6 mg / Kg. The adjuvant group was injected with the same volume of adjuvant three times a week. The results are as Figure 11 shown in Table 5. It was found that the subcutaneous tumors in nude mice caused by mTOR-activated cells were sensitive to the mTOR inhibitor rapamycin. The weight and volume of the subcutaneous tumors in the rapamycin injection group were smaller than those in the adjuvant group, and the difference was statistically significant. The tumor inhibition rate of rapamycin for the mTORmut group was 59.03%.

[0056] Table 5 Statistical results of tumor inhibition rate

[0057] Deletion of the tumor suppressor genes TSC1 / 2 upstream of mTOR can also cause activation of the mTOR signaling pathway. However, deletion of the TSC1 / 2 genes will negatively feedback inhibit the expression of p-AKT protein by upregulating the expression of the mTOR gene. This is the reason why the inactivation mutation of the TSC1 or TSC2 tumor suppressor gene causes tuberous sclerosis, a 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). However, for cells with autoactivation of mTOR, the present invention found that the expression of p-AKT protein is upregulated, which is different from the mechanism of tuberous sclerosis caused by TSC1 / 2 deletion, providing a reliable and stable cell model for the study of mTOR autoactivation diseases ( Figure 12 ).

[0058] Thus, the present invention mutated serine (Serine, S) at position 2215 of the mTOR protein to tyrosine (Tyrosine, Y) to construct a transgenic mouse model with the mTORS2215Y activation mutation. Using the transgenic mouse model, an immortalized mouse embryonic fibroblast cell line was constructed, so that the relevant mechanisms of mTOR autoactivation mutation can be studied at the cellular level, and relevant therapeutic drugs can be screened.

[0059] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of mammalian target of rapamycin (mTOR) auto-activation, characterized in that, Comprising the following steps: After connecting the insertion sequence with the homologous arms, construct it onto a homologous recombination vector to obtain a homologous recombination targeting vector; Transfer the homologous recombination targeting vector into ES cells to construct the heterozygous mTORS2215Y mutant mouse mTORS2215Y f / + ; The heterozygous mTOR S2215Y mutant mice mTOR S2215Y f / + were backcrossed after being crossed with wild-type mice to screen for homozygous mTOR S2215Y mutant mice mTOR S2215Y f / f , and the mouse model with self-activation of mammalian target of rapamycin was obtained; The nucleotide sequence of the insertion sequence is as shown in SEQ ID NO:

1.

2. The construction method according to claim 1, characterized in that 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.

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

4. Use of the mouse model with constitutive activation of mammalian target of rapamycin obtained by the construction method according to any one of claims 1 to 3 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.

5. A method for constructing a mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin, characterized in that, Comprising the following steps: The mouse model with self-activated mammalian target of rapamycin obtained by using the construction method described in any one of claims 1 to 3 is backcrossed, and fibroblasts are extracted to obtain fibroblasts mTORS2215Y f / f Mefs; Fibroblasts mTORS2215Y f / f After the Mefs were passaged to the 4th generation, they were mixed with lentivirus containing SV40 large T to obtain immortalized fibroblasts mTORS2215Y f / f Mefs; Immortalize the P6 generation of immortalized fibroblasts mTORS2215Y f / f Mix Mefs with cre adenovirus to obtain the mouse embryonic fibroblast cell line with self-activated mammalian target of rapamycin 6. The construction method according to claim 5, wherein The fibroblasts are taken from a mouse embryo, and the mouse embryo is the mouse embryo at 13.5 days after plugging detected after backcrossing of the mouse model with constitutive activation of mammalian target of rapamycin.

7. The construction method according to claim 5, characterized in that The multiplicity of infection (MOI) of the lentivirus containing SV40 large T is 2, and the mixing time of the lentivirus containing SV40 large T is 48 h.

8. The construction method according to claim 5, characterized in that The dosage of the Cre adenovirus is 1×10 10 pfu / mL, and the mixing time of the Cre adenovirus is 48 h.

9. Mouse embryonic fibroblast cell line with constitutive activation of mammalian target of rapamycin obtained by the construction method according to any one of claims 5 to 8.

10. Use of the mouse embryonic fibroblast cell line with constitutive activation of mammalian target of rapamycin obtained by the construction method according to any one of claims 5 to 8 or the mouse embryonic fibroblast cell line with constitutive activation of mammalian target of rapamycin according to claim 9 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.

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