WO2017143894A1 - Souris immunodéficiente, procédé de préparation correspondant et application correspondante - Google Patents

Souris immunodéficiente, procédé de préparation correspondant et application correspondante Download PDF

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WO2017143894A1
WO2017143894A1 PCT/CN2017/071697 CN2017071697W WO2017143894A1 WO 2017143894 A1 WO2017143894 A1 WO 2017143894A1 CN 2017071697 W CN2017071697 W CN 2017071697W WO 2017143894 A1 WO2017143894 A1 WO 2017143894A1
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mouse
mice
gene
nsi
mmol
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Chinese (zh)
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李鹏
蒋治武
魏新茹
李柏衡
林思妙
王素娜
姚瑶
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深圳市体内生物医药科技有限公司
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Priority to CN201911347317.1A priority Critical patent/CN110951787B/zh
Priority to CN201780000020.0A priority patent/CN106661593A/zh
Publication of WO2017143894A1 publication Critical patent/WO2017143894A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • the invention belongs to the technical field of animal genetic engineering, and particularly relates to an immunodeficient mouse, a preparation method thereof and application thereof.
  • the liver plays a strategic role in the body's function of detoxification and metabolism of foreign compounds. Due to the low cost of breeding, rapid reproduction, short experimental period, clear genetic background, and high experimental repeatability, the researchers used mice to construct various mouse liver disease models, such as liver injury, viral hepatitis, and liver fiber. Liver disease, liver cancer, etc., for liver disease research. However, these models are difficult to compensate for the large species differences between humans and mice (for example, many liver metabolic enzymes are species-specific, and some human liver viruses such as HBV, HCV, etc. cannot infect mouse liver cells). Outcomes or therapeutic drugs cannot reproduce the same effect on the human body. The in vitro culture of human liver cells is limited to the survival of human primary cells in vitro, cryopreservation techniques, and the inability to reproduce organ coordination in vivo.
  • liver-humanized mouse study A humanized mouse model of the liver, which transplants and reconstructs human liver tissue or organs in immunodeficient mice that can induce liver damage.
  • controlled inducible mouse liver cell damage provides sufficient space for the survival, proliferation and development of transplanted human liver cells.
  • TK-NOG Thymidine Kinase, thymidine kinase-deficient NOD-Scid IL2rg-/-
  • TK-NOG Thymidine Kinase, thymidine kinase-deficient NOD-Scid IL2rg-/-
  • uPA-SCID urokinase-type Plasminogen Activator
  • immunodeficient mice Mercer et al., 2001
  • FRG Fah-/- Rag2-/-il2 ⁇ c-/-
  • FRGN Fah-/-Rag2-/-il2 ⁇ c-/-NOD
  • TK transgenic male mice are infertile and can only pass heterozygous female and wild-type male mice without homozygous mouse strains, while uPA mice have very high requirements for donor liver cells, and uPA gene deletion can cause Rapid and severe liver damage with uncontrollable selectivity.
  • Liver-induced injury mice based on Fah knockout, such as FRG and FRGN are the most widely used, controllable, and highly portable liver model tools, and can be used to protect liver drugs NTBC (2-nitro-4).
  • NTBC (2-nitro-4).
  • -Trifluorotoluene-1,3 cyclohexanedione controls Fah deletion-induced liver damage (Grompe et al., 1995;).
  • Fah knockout mice based on the NOD-scid IL2rg-/- immunodeficiency background will be the best humanized mouse model tool for liver disease.
  • researchers have encountered a series of difficulties in exploring the development of this genotype mouse. Due to the complex genetic background of NOD (non-obese diabetic) mice and spontaneous non-obese diabetes, gene knockout in NOD mice may induce lethal or diabetic disease (Baxter AG et al., 1995; Nichols J et Al., 2009;).
  • scid mice have defects in Prkdc gene, partial loss of DNA repair ability, affecting gene repair after off-targeting of gene knockout tools, and increasing mortality in knockout mice.
  • NOD-scid Fah-/- mice died rapidly after NTBC (liver protection drug) discontinuation, and mice died (Blunt, T. et al., 1996).
  • NOD-scid IL2rg has a higher genetic background defect than NOD and NOD-scid mice.
  • mice with missing immune cells such as B, T, NK, etc.
  • these mice have hair.
  • fluorescent markers especially fluorescent proteins
  • the present invention provides an immunodeficient mouse, which is better than the existing NSI or other third generation immunodeficient mice, and a preparation method and application thereof. Effect.
  • the present invention provides a method of gene knockout using NOD-Scid IL2rg-/- immunodeficient mice (NSI mice), which knocks out the Fah gene or the Foxn1 gene.
  • NSIF breeding NSIF
  • NOD-scid IL2rg-/-Fah-/- is obtained by performing Fah gene knockout in the background of immunodeficient mouse NOD-Scid IL2rg-/-, which is currently recognized as having the highest degree of immunodeficiency.
  • a mouse strain, and a novel hepatic humanized mouse model with high chimerism and high success rate was constructed using NSIF mice.
  • the model can be used for the evaluation of the efficacy of scientific research and treatment methods (such as chemical drugs, biological agents, etc.) of human liver diseases (such as fatty liver, liver damage, hepatitis, liver cancer, etc.), as well as for drug catabolism and toxicity assessment.
  • scientific research and treatment methods such as chemical drugs, biological agents, etc.
  • human liver diseases such as fatty liver, liver damage, hepatitis, liver cancer, etc.
  • a highly immunodeficient mouse NSEN having a hair development defect is obtained by performing Foxn1 gene knockout in the background of immunodeficient mouse NOD-Scid IL2rg-/- which is currently considered to have the highest degree of immunodeficiency.
  • NOD-Scid IL2rg-/-Foxn1-/- is obtained by performing Foxn1 gene knockout in the background of immunodeficient mouse NOD-Scid IL2rg-/- which is currently considered to have the highest degree of immunodeficiency.
  • the nucleotide target sequence of the Fah gene is represented by SEQ ID NO. 1
  • the nucleotide target sequence of the Foxn1 gene is represented by SEQ ID NO.
  • the nucleotide sequence is as follows:
  • Fah aagctgcatggaagg (SEQ ID NO. 1);
  • Foxn1 ggaagtgcctcttgtagggg (SEQ ID NO. 2).
  • the invention provides a method of knocking out a Fah gene according to the first aspect, comprising the steps of:
  • step (4) The chimeric or hybrid NSIF immunodeficient mice obtained in step (4) are crossed with NSI mice to obtain more hybrid NSIF immunodeficient mice, and hybrid NSIF immunodeficient mice are crossed to obtain NSIF homozygous immunity. Defective mice.
  • the construction of the TALEN plasmid described in the step (1) comprises the steps of: obtaining a TALEN left arm recognition binding sequence and a TALEN right arm recognition binding sequence according to a target sequence of the Fah gene, respectively, designing a coding TALEN left arm and a right Arm repeats, then ligating repeats to TALEN expression On the vector, pCAG-TALEN L (left arm)-X-pA and pCAG-TALEN R (right arm)-X-pA plasmids were obtained.
  • the TALEN left arm recognition binding sequence is SEQ ID NO. 3, and the TALEN right arm recognition binding sequence is SEQ ID NO. 4;
  • TALEN left arm recognition binding sequence 5-aacttcatgggtctgggtc-3 (SEQ ID NO. 3);
  • TALEN right arm recognizes the binding sequence: 5-aaggatgctcttgcct-3 (SEQ ID NO. 4).
  • the TALEN mRNA is obtained according to the procedures of the kits M MESSAGE SP6 (Ambion) and E. coli Poly (A) Polymerase (NEB).
  • the concentration of TALEN mRNA injected into the cytoplasm of the fertilized egg of the NSI mouse according to the step (2) is 10-200 ng/ ⁇ L, for example, 10 ng/ ⁇ L, 11 ng/ ⁇ L, 12 ng/ ⁇ L, 13 ng/ ⁇ L, 15 ng.
  • the medium for the embryonic operation of the fertilized egg of the NSI mouse knocking out the Fah gene is 10-40 mmol/L of HEPES, pH 7.0-8, the non-essential amino acid is 0.05-1 mmol/L, and the essential amino acid is 0.1- 2 mmol/L, preferably 20 mmol/L of HEPES, pH 7.4-7.8, 0.1 mmol/L of non-essential amino acids, and 0.1-0.6 mmol/L of essential amino acids.
  • the medium for the mouse embryo culture in which the Fah gene is knocked out is a pyruvic acid concentration of 0.1 to 2 mmol/L, a glutamine concentration of 0.5 to 3 mmol/L, a glucose concentration of 0.01 to 1 mmol/L, preferably a pyruvic acid concentration. 0.35 mmol/L, glutamine concentration 1 mmol/L, glucose concentration 0.1 mmol/L.
  • the number of fertilized eggs transplanted into the uterus of the pseudopregnant mouse according to the step (2) is 10-20 pieces.
  • it may be 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19 or 20, preferably 10-15.
  • the pseudopregnant mouse is any one of a NOD mouse, a NOD-SCID mouse or an NSI mouse.
  • the method of knocking out the Foxn1 gene further comprises selecting a surrogate mouse for lactating newborn mice.
  • the surrogate mouse is an ICR mouse.
  • the present invention provides the NSIF mouse obtained by knocking out the Fah gene by the method of gene knockout according to the second aspect.
  • the present invention provides an immunodeficient mouse model further genetically engineered based on the NSIF mouse of the third aspect.
  • the present invention provides the use of a mouse model according to the third aspect or the fourth aspect as a model mouse for pathological and physiological studies of the human body, preferably as a model mouse for liver disease research and/or The use of liver-humanized model mice.
  • the present invention provides a method of knocking out the Foxn1 gene, comprising the steps of:
  • step (3) The chimeric or hybrid NSIN immunodeficient mice obtained in step (3) were crossed with NSI mice to obtain more hybrid NSIN immunodeficient mice, and hybrid NSIN immunodeficient mice were crossed to obtain NSIN homozygous immunity. Defective mice.
  • the obtained mRNA is injected into the mouse fertilized egg cells by pronuclear injection, and the various embryo culture mediums during the pronuclear injection process are adapted to the NSI embryos, thereby improving the efficiency of the embryonic pronucleus injection and the recovery of the embryo after injection.
  • the step of constructing a Foxn1 gene knockout recombinant vector comprises: obtaining a linearized guide DNA (L-gDNA) by a primer, and then ligating the linearized L-pT7 vector by a DNA ligase to obtain a pT7-gDNA integrity. Carrier.
  • nucleotide sequence of the primer is shown in SEQ ID NO. 5-6;
  • the nucleotide sequence is as follows:
  • SEQ ID NO. 5 5'-ATAGGN ggaagtgcctcttgtagggg GT-3';
  • SEQ ID NO. 6 5'-TAAAACN cccctacaagaggcacttccG-3';
  • the N may represent any one of A, T, G or C.
  • the in vitro transcription of the gRNA comprises the steps of: amplifying a gDNA gene fragment by a primer using a pT7-gDNA intact vector as a template, and performing in vitro transcription to obtain a gRNA.
  • nucleotide sequence of the primer is shown in SEQ ID NO. 7-8;
  • SEQ ID NO. 7 5'-GAAATTAATACGACTCACTATA-3';
  • SEQ ID NO. 8 5'-AAAAAAAGCACCGACTCGGTGCCAC-3'.
  • the in vitro transcription of Cas9 mRNA according to step (2) comprises the steps of linearizing the pcDNA3.3-hCas9 vector, recovering the linearized pcDNA3.3-hCas9 vector as a template for in vitro transcription, and using the SP6 polymerase promoter at Cas9 mRNA was transfected in vitro, and capped Cas9 mRNA was recovered by Licl method, and polyA was added, and purified and stored.
  • the concentration of gRNA and Cas9 mRNA injected into the cytoplasm of the fertilized egg of the NSI mouse according to the step (3) is 10-200 ng/ ⁇ L, for example, 10 ng/ ⁇ L, 11 ng/ ⁇ L, 12 ng/ ⁇ L, 13 ng/ ⁇ L, 15 ng/ ⁇ L, 18 ng/ ⁇ L, 20 ng/ ⁇ L, 25 ng/ ⁇ L, 30 ng/ ⁇ L, 35 ng/ ⁇ L, 40 Ng/ ⁇ L, 45 ng/ ⁇ L, 50 ng/ ⁇ L, 55 ng/ ⁇ L, 60 ng/ ⁇ L, 65 ng/ ⁇ L, 70 ng/ ⁇ L, 75 ng/ ⁇ L, 80 ng/ ⁇ L, 85 ng/ ⁇ L, 90 ng/ ⁇ L, 100 ng/ ⁇ L, 110 ng/ ⁇ L, 120 ng/ ⁇ L, 130 ng/ ⁇ L, 140 ng/ ⁇ L, 150 ng/ ⁇ L, 160 ng/ ⁇ L, 170 ng/ ⁇ L, 100
  • the embryo of the NSI mouse knockout Foxn1 gene is used for embryo manipulation of 10-40 mmol/L HEPES, pH 7.0-8, non-essential amino acids 0.05-1 mmol/L, and essential amino acids 0.1-2 mmol. /L, preferably 20 mmol/L of HEPES, pH 7.4-7.8, non-essential amino acid 0.1 mmol/L, and essential amino acid 0.1-0.6 mmol/L.
  • the medium for culturing the mouse embryo of the Foxn1 gene has a pyruvic acid concentration of 0.1 to 2 mmol/L, a glutamine concentration of 0.5 to 3 mmol/L, a glucose concentration of 0.01 to 1 mmol/L, preferably a pyruvic acid concentration. 0.35 mmol/L, glutamine concentration 1 mmol/L, glucose concentration 0.1 mmol/L.
  • the number of fertilized eggs transplanted into the uterus of the pseudopregnant mouse according to the step (3) is 10-20 pieces, for example, 10 pieces, 11 pieces, 12 pieces, 13 pieces, 14 pieces, 15 pieces, 16 pieces, and 16 pieces. 17, 17 pieces, 19 pieces, 19 pieces or 20 pieces, preferably 10-15 pieces.
  • the pseudopregnant mouse is any one of a NOD mouse, a NOD-SCID mouse or an NSI mouse.
  • the method of knocking out the Foxn1 gene further comprises selecting a surrogate mouse for lactating newborn mice.
  • the surrogate mouse is an ICR mouse.
  • mice that have knocked out the Foxn1 and Fah genes use NSI female mice as surrogate mothers, and ICR mothers with at least one production experience are used as surrogate mothers.
  • the use of NSI female mice as surrogate mothers is due to the growth environment of the genotype and the in vivo microenvironment, which is more suitable for the growth of the defective mice, which can improve the survival rate; and the use of ICR mothers as surrogate mothers can improve the survival rate.
  • Breastfeeding rate is due to the growth environment of the genotype and the in vivo microenvironment, which is more suitable for the growth of the defective mice, which can improve the survival rate; and the use of ICR mothers as surrogate mothers can improve the survival rate.
  • the present invention provides the NSIN mouse obtained by knocking out the Foxn1 gene by the method of gene knockout according to the first aspect.
  • the transplantation rate of the transplanted tumor, the survival and growth of the xenogeneic cells of the NSIN mouse are obviously superior to those of other immunodeficient mice, and the degree of immunodeficiency of the NSIN mouse is high, which can be regarded as the optimal tumor disease research.
  • Model mice are obviously superior to those of other immunodeficient mice, and the degree of immunodeficiency of the NSIN mouse is high, which can be regarded as the optimal tumor disease research.
  • the present invention provides an immunodeficient mouse model further genetically engineered based on the NSIN mouse of the seventh aspect.
  • the present invention provides the use of the mouse model according to the seventh aspect or the eighth aspect as a model mouse for human pathological and physiological studies, preferably as a model mouse for tumor disease research.
  • the present invention provides the mouse obtained by knocking out the Fah gene and the Foxn1 gene by the method of gene knockout according to the first aspect.
  • the culture medium used for the embryonic operation such as transplanting fertilized eggs in the Foxn1 and Fah gene mice is 20 mmol/L of HEPES, pH 7.4-7.8, and the non-essential amino acid is 0.1 mmol/L.
  • the amino acid is from 0.1 to 0.6 mmol/L.
  • the medium used for the mouse embryo culture in the knockout Foxn1 and Fah gene mice was a pyruvic acid concentration of 0.35 mmol/L, a glutamine concentration of 1 mmol/L, and a glucose concentration of 0.1 mmol/L.
  • the present invention has the following beneficial effects:
  • the immunodeficient mouse with the highest degree of immunodeficiency in the present invention is subjected to Foxn1 gene knockout in the background of NOD-Scid IL2rg-/-, and a highly immunodeficient mouse NSN (NOD-Scid) having a hair development defect (almost no hair) is obtained.
  • NOD-Scid highly immunodeficient mouse NSN
  • the mouse has no body hair, it is easier to observe the cell growth, development and migration of the mouse, and it is easy to observe and size the solid tumor, and prevent the mice with high immunodeficiency Probabilistic thymic rebirth events that occur with ageing;
  • the present invention performs Fah gene knockout in the context of NOD-Scid IL2rg-/- in immunodeficient mice, Obtained a sustainable breeding NSIF (NOD-scid IL2rg-/-Fah-/-) mouse strain, Fah deletion can induce liver failure, and immune cells participate in subsequent liver failure, the higher the degree of NSIF immunodeficiency, can alleviate liver failure
  • NSIF novel liver humanized mouse model with high chimerism and high success rate was constructed using NSIF mice, which can be used for human liver diseases (such as fatty liver, liver injury, hepatitis, liver cancer, etc.). Evaluation of the efficacy of scientific research and therapeutic methods (eg, chemicals, biological agents, etc.), as well as catabolic and toxicity assessments for therapeutic methods;
  • the present invention uses NSI mice to surrogate, and ICR mice are cultured to culture NSN and NSIF mice, thereby improving the survival rate and lactation rate of the defective mice, and the present invention will be obtained by pronuclear injection.
  • the mRNA is injected into the fertilized egg, and the NSI embryos are adjusted by various embryo culture media in the pronuclear injection to improve the efficiency of pronuclear injection and recovery of the embryo after injection.
  • 1 is a result of PCR digestion of the NSIN mouse gene of the present invention, wherein WT is wild type, Foxn1 +/- is heterozygous, and Foxn1-/- is homozygous;
  • FIG. 2 is a result of PCR digestion of the NSIF mouse gene of the present invention, wherein WT is wild type, Fah+/- is heterozygous, and Fah-/- is homozygous;
  • Figure 3 is a result of alignment of the NSIF mouse sequencing results of the present invention on NCBI;
  • Figure 4 is a diagram showing the results of Western Blot of detecting Fah protein expression in NSIF mice with liver injury according to the present invention
  • Fig. 5 is a liver pathological section of the NSIF mouse of the present invention, wherein Fig. 5(A) shows liver pathological sections after NSIF mice stop using NTBC drugs, and Fig. 5(B) shows liver pathology of NSIF mice after normal use of NTBC drugs. slice;
  • Figure 6 is a graph showing the expression levels of alanine aminotransferase and aspartate aminotransferase after cessation of NTBC drugs in NSIF mice of the present invention
  • Figure 7 is a graph showing the expression level of glucose after cessation of NTBC drugs in NSIF mice of the present invention.
  • Figure 8 is a graph showing changes in body weight of livers of NSIF mice of the present invention after transplantation of liver transplanted/non-transplanted C57BL/6 mice, and discontinuation of NTBC drugs, wherein NISF-NTBC+liver is a NSIF mouse transplanted with liver of C57BL/6 mice.
  • NSIF-NTBC is a non-transplanted liver NSIF mouse;
  • Figure 9 is a graph showing the expression levels of alanine aminotransferase and aspartate aminotransferase in the liver of the transplanted/non-transplanted C57BL/6 mouse of the NSIF mouse of the present invention after stopping the use of the NTBC drug, wherein the NISF-NTBC+liver is NSIF mice transplanted with liver of C57BL/6 mice, NSIF-NTBC were mice without transplanted liver;
  • Figure 10 is a graph showing the expression level of glucose in the liver of the NSIF mouse of the present invention after transplantation/non-transplantation of C57BL/6 mice, wherein the NISF-NTBC+liver is a liver transplanted with C57BL/6 mice, NSIF-NTBC a mouse that has not been transplanted with liver;
  • Figure 11 is a schematic view showing the structure of the Fah target site and the TALEN left and right arms;
  • Figure 12 is a comparison of the reconstitution efficiency of peripheral blood NALM6-GFP in mice after transplantation of 1 ⁇ 10 4 NALM6-GFP cells into NOG, NSI and NSIN mice, respectively;
  • Figure 13 is a comparison of the reconstitution efficiency of peripheral blood NALM6-GFP in mice after transplantation of 1 ⁇ 10 5 NALM6-GFP cells into NOG, NSI and NSIN mice, respectively;
  • Figure 14 is a comparison of the reconstitution efficiency of peripheral blood NALM6-GFP in mice after transplantation of 1 ⁇ 10 6 NALM6-GFP cells into NOG, NSI and NSIN mice, respectively;
  • Figure 15 is a comparison of the weight of subcutaneous tumor tissue in mice after transplantation of 1 ⁇ 10 4 A549 cells into NOG, NSI, and NSIN mice for 30 days;
  • Figure 16 is a comparison of the weight of subcutaneous tumor tissue in mice after transplantation of 1 ⁇ 10 5 A549 cells into NOG, NSI, and NSIN mice for 30 days;
  • Figure 17 is a comparison of the weight of subcutaneous tumor tissue in mice after transplantation of 1 ⁇ 10 6 A549 cells into NOG, NSI, and NSIN mice for 30 days.
  • All animals of the present invention are raised and propagated at the SPF (Specific Pathogen Free) level experimental animal center.
  • the NSI mouse of the present invention genotype: NOD-scid IL2rg-/-, authorized patent number: ZL201310229629.9.
  • Target selection Using the ZiFiT Targeter Version website to design a GGN (17-18) NGG (N is an arbitrary base) sequence following the Cas9 knockout target requires a Cas9 knockout target site and pass Ensembl/NCBI The "Blast" search function of the website determines that the target site is a single site in the genome;
  • Target site sequence ggaagtgcctcttgtagggg (SEQ ID NO. 1)
  • Target confirmation According to the genome of the target cell, a high specific primer for amplifying the target site is designed, and a target site fragment is obtained by PCR amplification; the only restriction endonuclease of the amplified fragment is selected in the target site for enzymatic digestion. After electrophoresis identification; after the enzyme digestion is correctly identified, the PCR amplification products are sent to the sequencing identification; the specificity of the primers for identification and the feasibility of enzyme digestion and sequencing identification are confirmed by enzyme digestion and sequencing identification;
  • L-pT7 and L-gDNA are ligated into the complete vector pT7-gDNA by DNA ligase (such as Takara Solution I), transformed, plated, picked monoclonal, shaken, plasmid DNA extracted, restriction enzyme identification, plasmid Sequencing, screening and sequencing the correct plasmid for use;
  • DNA ligase such as Takara Solution I
  • T7-S primer sequence 5'-GAAATTAATACGACTCACTATA-3' (SEQ ID NO. 5)
  • Tracr-Rev primer sequence 5'-AAAAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO. 6)
  • the capped Cas9 mRNA was transcribed in vitro with mMESSAGE mMACHINE T7Kit according to the following system and procedure, electrophoresis, running gel, and capped Cas9 mRNA.
  • the reaction system was configured according to the following system, and polyA was added to the capped Cas9 mRNA to obtain a stable and more efficient RNA;
  • NSI male and female sperm that provide sperm NSI maternal and parental mice (the mother and father of the NSI male and female rats that provide sperm) are caged and the parental mouse is divided. Out, put in ICR mother rats with at least one production experience to ensure adequate feeding of NSI maternal rats;
  • surrogate NSI rats After the NSI maternal and maternal rats of the surrogate mother are seen in the cage, the father rats are separated and placed in an ICR mother with at least one production experience. The surrogate mother is out. 7 days after birth, the same litter males were taken out to ensure sufficient supply of nutrients during the lactation period of the surrogate mothers;
  • mice genotype is identified by genotyping (PCR amplification and sequencing) to obtain hybrid NSIN (NOD-scid IL2rg-/-Foxn1-/-) immunodeficient mice, which is further reduced by NSI.
  • NSIN NOD-scid IL2rg-/-Foxn1-/-
  • the NSIN homozygote was obtained by hybridization of the mouse, and the PCR results are shown in Figure 1.
  • the homozygous NSIN mouse is defective in the Foxn1 gene.
  • TALEN target sequence 5-aagctgcatggaagg-3;
  • TALEN left arm recognition binding sequence 5-aacttcatgggtctgggtc-3;
  • TALEN right arm recognition binding sequence 5-aaggatgctcttgcct-3;
  • TALEN left arm AACTTCATGGGTCTGGGTCAAG;
  • TALEN right arm AAGGATGCTCTTGCCTCCT;
  • the single-strand annealing (SSA) method was used for the TALEN activity in vitro.
  • the reporter gene in the SSA reporter vector was the luciferase gene, and the promoter was CMV.
  • the method was to transfect 200 ng TALENs expression plasmid, 50 ng SSA reporter plasmid and 10 ng Renilla plasmid into 24-well plate 293T cells. After 1 day, the transfected cells were collected and treated with Luciferase Cell Lysis Buffer (NEB) to detect luciferase activity and predict TALEN. Cutting activity;
  • TALENs plasmid requires embryonic cytoplasmic injection after in vitro transcription synthesis of TARNA encoding TALEN and poly(A) at the end.
  • the in vitro transcription and polyadenylation of TALEN m RNA was carried out according to the procedures of the kits M MESSAGE SP6 (Ambion) and E. coli Poly (A) Polymerase (NEB). The method is as follows:
  • TALEN m RNA was precipitated and recovered, and the in vitro transcribed Cas9 RNA was recovered from the transcription system by using the mirVana TM miRNA Isolation Kit kit (Ambion), dissolved in 10-20 ⁇ L of enzyme-free water, and stored at -80 ° C until use.
  • NSI male and female sperm that provide sperm NSI maternal and parental mice (the mother and father of the NSI male and female rats that provide sperm) are caged and the parental mouse is divided. Out, put ICR mothers with at least one production experience to ensure adequate feeding of NSI maternal rats.
  • NSI maternal and maternal rats in the surrogate mothers were found to be tied together, and the parent mice were separated and placed in an ICR mother with at least one production experience. The surrogate mother was born. After the next 7 days, the same litter males were taken out to ensure that the lactating mothers had sufficient nutrient supply during lactation.
  • mice genotype was identified by genotyping (PCR amplification and sequencing) to obtain hybrid NSIF (NOD-scid IL2rg-/-Fah-/-) immunodeficient mice, and further The NSIF homozygote was obtained by hybridization with NSI mice, and the NSIF sequencing results were compared on NCBI. The results are shown in Fig. 3, and the results of PCR electrophoresis are shown in Fig. 2;
  • NTBC final concentration: 7.5mg/L
  • NTBC final concentration: 7.5mg/L
  • NTBC final concentration: 7.5mg/L
  • 120ul is needed one week before the mother gives birth. /only; after the mother is born, the rats less than 4 weeks old need to be injected with NTBC 10-20uL per day, and the lactating mother is injected with NTBC 120uL/only;
  • Fah protein in the liver of NSIF mice was detected by Western Blot (NSI mice were used as positive control). As shown in Fig. 4, NSI expressed Fah protein, and Fah protein was not expressed in NSIF mice, which proved that Fah gene knockout was successful.
  • NTBC NSIF mice were weighed and recorded daily, and the effects of NTBC on the body weight changes of NSIF mice were removed by plotting the body weight change curve compared with NSIF mice drinking NTBC acidic water.
  • NSIF mice whose birth dates differ by no more than one week and of the same sex (born with the same fetus), divided into two groups (3 in each group), one group being NSIF-NTBC+Liver (ie NSIF mice) After transplantation of C57BL/6 mouse liver cells, the NTBC drug was gradually stopped.
  • One group was NSIF-NTBC (ie, NSIF mice that did not transplant C57BL/6 mouse liver cells, and NTBC gradually evolved with the NSIF-NTBC+Liver experimental group). Stop the drug);
  • Each NSIF-NTBC+Liver mouse was injected with 100 ⁇ L of C57BL/6 mouse single liver cell suspension in step 2 by intravenous injection, that is, each mouse was injected with 2 ⁇ 10 6 cells;
  • mice in both groups were stopped from taking NTBC and daily
  • the experimental mice were weighed and recorded weight as shown in Figure 8;
  • ALT Alanine Aminotransferase
  • AST Aspartate Aminotransferase
  • glucose Glucose
  • NSIF mice that did not transplant C57BL/6 mouse liver cells gradually lost weight until they died, while NSIF mice transplanted with C57BL/6 mouse liver cells gradually lost weight for 33 days. restore. It is indicated that mouse liver cells of different strains transplanted can grow in mice and replace the liver function of recipient mice.
  • Example 7 Construction of a solid tumor humanized mouse model using NSIN
  • NALM6-GFP cells human B lymphoblastic acute leukemia BALL cell line, labeled with green fluorescent protein
  • NSIN human B lymphoblastic acute leukemia BALL cell line, labeled with green fluorescent protein
  • NSI human B lymphoblastic acute leukemia BALL cell line, labeled with green fluorescent protein
  • NOG NOD.Cg-Prkdc scid IL2rg tmlSug / JicCrl, Japan CIEA / IVS company
  • Figure 12-14 shows that the in vivo environment of NSIN immunodeficient mice is more suitable for the survival and growth of xenogeneic (hematoma, normal blood) cells; When the cells are small, the transplantation efficiency of NSIN mice is significantly higher than that of NSI and NOG mice; preliminary conclusions can be drawn that the order of immunodeficiency of the three mice is NSIN>NSI>NOG.
  • Example 8 Construction of a solid tumor humanized mouse model using NSIN
  • A549 cells human lung adenocarcinoma cell line
  • NSIN subcutaneous injection
  • NSI NOD/SCID IL2rg-/-
  • NOG NOD.Cg-PrkdcscidIL2rgtm1Sug/JicCrl, Japan CIEA/IVS
  • Figure 15-17 shows that the in vivo environment of NSIN immunodeficient mice is more suitable for the survival and growth of xenogeneic (solid tumor, non-blood normal cells) cells; especially in When the cells were few, the transplantation efficiency of NSIN mice was significantly higher than that of NSI and NOG mice. It was further verified that the order of immunodeficiency of the three mice was NSIN>NSI>NOG.

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Abstract

La présente invention concerne une souris immunodéficiente, un procédé de préparation correspondant et une application correspondante. Le procédé de préparation concerne la désactivation du gène Foxnl ou du gène FAH d'une souris immunodéficiente NOD-Scid IL2rg-/- (souris NSI). Par la désactivation du gène FAH d'une souris NSI, on obtient une souris NSIF. La souris NSIF peut être utilisée pour construire efficacement un nouveau modèle de souris humanisé qui peut être utilisé pour les études physiologiques et pathologiques du foie. Par la désactivation du gène Foxnl d'une souris NSI, on obtient une souris NSIN. La souris NSIN est sans poils et présente en outre un système immunitaire déficient.
PCT/CN2017/071697 2016-02-25 2017-01-19 Souris immunodéficiente, procédé de préparation correspondant et application correspondante WO2017143894A1 (fr)

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CN114763557A (zh) * 2021-01-13 2022-07-19 北京市农林科学院 Ddx5在抗病毒和调节免疫反应中的应用
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CN112522202B (zh) * 2020-09-23 2022-04-05 南京启真基因工程有限公司 制备addi四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法及其专用试剂盒
CN112237637A (zh) * 2020-10-10 2021-01-19 广州市天河诺亚生物工程有限公司 一种基于免疫缺陷小鼠脑瘫模型的细胞治疗研究方法
CN112243949A (zh) * 2020-11-09 2021-01-22 广州市天河诺亚生物工程有限公司 一种脑瘫小鼠动物模型的构建及评价方法
CN113106101B (zh) * 2021-05-11 2023-04-07 广州欣意生物技术有限公司 一种nod遗传背景双基因缺陷小鼠模型的制备方法及应用
WO2023077360A1 (fr) * 2021-11-04 2023-05-11 云南农业大学 Procédé de construction d'un modèle porcin double souffrant d'immunodéficience sévère et de lésions hépatiques et application

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