WO2018228534A1 - Procédé de préparation de rats immunodéficients et utilisation associée - Google Patents

Procédé de préparation de rats immunodéficients et utilisation associée Download PDF

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WO2018228534A1
WO2018228534A1 PCT/CN2018/091503 CN2018091503W WO2018228534A1 WO 2018228534 A1 WO2018228534 A1 WO 2018228534A1 CN 2018091503 W CN2018091503 W CN 2018091503W WO 2018228534 A1 WO2018228534 A1 WO 2018228534A1
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seq
gene
rat
sgrna
liver
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惠利健
张鲁狄
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中国科学院上海生命科学研究院
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Definitions

  • the present invention is in the field of biology, and more particularly, the present invention relates to methods of preparing immunodeficient rats and their use.
  • Liver disease is one of the most important diseases that threaten human health.
  • hepatitis virus-induced hepatitis, cirrhosis and liver cancer, and malaria caused by malaria parasites affect human health and cause a serious economic burden.
  • the pathogenesis and treatment of liver diseases is extremely complicated, and human liver cells rapidly lose liver characteristics after in vitro culture. Therefore, in addition to the cellular level, it is more necessary to conduct related research from the body level by means of animal models.
  • hepatopathic pathogens are highly host-specific and can only infect human and a few high-level primates (such as chimpanzees, etc.), making it impossible to conduct related liver disease studies in traditional animal models such as mice.
  • Liver humanized animals can be established by reconstituting the liver of an immunodeficient animal after transplantation of human liver cells.
  • a liver humanized animal is constructed by first isolating primary hepatocytes from a liver donor and then transplanting them into an immunodeficient animal model (such as uPA-SCID mice) with liver damage. After 6-8 weeks of colonization, human liver cells reconstituted the mouse liver. Based on the amount of human serum albumin secreted in the serum, a humanized mouse with a high integration ratio was determined for subsequent drug metabolism, safety, and hepatitis virus infection.
  • liver metabolic enzymes and transporter expression and function in liver-humanized mice have been shown to be nearly identical to human liver.
  • HBV drug non-auritoin failed in clinical stage II due to severe liver toxicity resulting in 5 deaths.
  • Treatment of liver-derived mice with non-uridine results in liver damage at low doses and can produce all clinical signs and pathologies in patients including severe impairment of liver function, fatty liver and hepatocyte mitochondrial destruction. Therefore, liver humanized animals have application prospects in pre-clinical drug metabolism, drug interaction and safety evaluation.
  • Alb-uPA mice This type of mouse will have severe and even fatal intestinal and abdominal bleeding after birth.
  • Joerg Petersen Laboratories Alb-uPA mice were mated with Rag2 -/- immunodeficient mice to obtain uPA/Rag2 -/- mice.
  • Liver-derived mice with a repopulation ratio (15%) were obtained for the first time by human liver cell spleen transplantation.
  • Katsutoshi Yoshizato Laboratories achieved a very high proportion (>90%) of human hepatocyte repopulation in uPA/SCID mice.
  • the current Alb-uPA mouse model has the following problems: First, the mortality of newborn mice is very high, and nearly half of the offspring die from abdominal or visceral hemorrhage within 4 days after delivery, and the window period of transplantation is short (need to be born) In the second 2 weeks, the transplant was performed. Second, only part of the surviving mice were carriers of the uPA gene, and some mice also had problems with transgene inactivation. Third, the transplanted human hepatocytes secreted complement to mice. Kidney function damage, further increasing mortality.
  • Fuarylacetoacetate hydrolase (FAH) knockout mice (Fah -/- mice) were established in 1993. Fah -/- mice accumulate succinyl in the liver due to inability to undergo complete tyrosine metabolism, resulting in the death of liver parenchymal cells. Fah -/- mice supplemented with NTBC in drinking water have no obvious difference in phenotype from wild-type mice, normal liver function and normal development and reproduction, but will have liver function within 4-6 weeks after stopping feeding NTBC. Death and death. Fah -/- mice have extensive and persistent liver damage, and their liver microenvironment is particularly suitable for the proliferation of transplanted cells.
  • Wild hepatocytes can almost completely reconstitute Fah -/- mice after spleen transplantation.
  • the liver over 90%
  • the recipient mice returned to normal liver function.
  • Fah -/- mice and Rag2 -/- IL2rg -/- immunodeficient mice were crossed, a three-knockout Fah -/- Rag2 -/- IL2rg -/- mouse (FRG mouse) was obtained.
  • FRG mice lack mature T, B cells and NK cells, and when transplanted with human liver cells, a high degree (>90%) of human hepatocytes can be repopulated.
  • Hiroshi Suemizu Laboratories used a new mouse model of inducible liver injury, Thymidine Kinase-Nod/SCID/IL2rg -/- (TK-NOG mice), to construct a high proportion (>90%) of liver-humanized mice.
  • TK-NOG mice Thymidine Kinase-Nod/SCID/IL2rg -/- mice
  • the Herpes simplex virus type 1thymidine kinase was specifically expressed in the liver using the Alb promoter.
  • the compound is harmless to normal cells, but is metabolized into toxic products in mouse hepatocytes expressing the TK gene to cause hepatocyte death, providing a repopulation space for transplanted human hepatocytes.
  • This mouse benefited from the extent of its immunodeficiency, and the average repopulation ratio of human hepatocytes was higher than that of Alb-uPA/SCID mice and FRG mice.
  • this immunodeficient mouse can only be propagated by artificial insemination, and the feeding environment is extremely clean and difficult to promote.
  • FKBP FK506-binding protein
  • Human hepatic progenitor cells and CD34 + hematopoietic stem cells were isolated from aborted fetal embryo liver, and the two groups of cells were injected into the liver of neonatal mice irradiated 1-5 days to establish humanized liver and immune system mice.
  • AFC8-hu HSC/Hep This mouse not only supports HCV viral infection, but also produces a human T cell mediated immune response. Moreover, it was first discovered that HCV infection can induce hepatitis and liver fibrosis in mice, accompanied by activation of stellate cells and expression of genes involved in human fibrogenesis.
  • the model in view of immune rejection, the model must use the embryonic liver of aborted fetus as an experimental material to obtain hepatocytes and hematopoietic stem cells from the same individual, and there are ethical problems.
  • the relatively low proportion of human hepatocyte repopulation (10-30%) and the relative immatureness of hepatic progenitors are defects in this model.
  • the rat As a medium-sized experimental animal, the rat is 10 times larger than the mouse, which is easy to operate. It can also provide more biological materials for experimental analysis. Rats are more closely related to humans in terms of physiology and pathology, and are more widely used. For testing drug treatment effects and toxicity tests. Therefore, rats have become a research hotspot in the research field of liver humanized animals. If liver-humanized rats are obtained, they will have more application prospects than mice, especially in the field of drug development. On the other hand, although liver-humanized mice can be used as bioreactors to produce human hepatocytes, only 8 to 15 ⁇ 10 7 hepatocytes can be produced per mouse, and the number is low.
  • a humanized rat liver can obtain human hepatocytes of the order of 10 9 to effectively alleviate the problem of lack of human liver cells. Therefore, liver-humanized rats are more suitable as bioreactors for human hepatocytes than mice. At the same time, one of the biggest costs of liver-humanized mouse construction is also human hepatocytes, resulting in a price of $3,500 per mouse. The provision of a large number of human hepatocytes by humanized rats in the liver will greatly reduce the construction cost of liver humanized animals and promote their wider application.
  • this model is mainly limited to the following two aspects: 1) Rats still retain T cells in Rag1 -/- , and NK cells are even more than wild type.
  • the use of immunosuppressive means can reduce immune rejection, but can not be completely removed, and the cost is extremely high; 2) the use of retrosine combined with liver transplantation model, the surgical procedure is complex, and the rate of repopulation of hepatocytes is relatively slow.
  • the bottleneck in the construction of liver-humanized rats is firstly the lack of an immunodeficient rat model that induces liver damage, followed by the lack of a technical system for human hepatocyte-transplanted rats.
  • Object of the present invention is to provide a Fah - / - Rag2 - / - IL2rg - / - rats preparation and use Fah - / - Rag2 - / - IL2rg - / - rat hepatocytes in vivo to achieve amplification and Construction Liver humanized rats.
  • a method for producing an immunodeficient rat comprising: disrupting a rat recombinant activating gene 2 (Rag2) and an interleukin 2 receptor ⁇ (IL2rg) gene; thereby obtaining an immunodeficient rat .
  • Rag2 rat recombinant activating gene 2
  • IL2rg interleukin 2 receptor ⁇
  • a method for producing a rat cell comprising: disrupting a rat recombinant activating gene 2 (Rag2) and an interleukin 2 receptor ⁇ (IL2rg) gene; preferably, the cell is Fertilized egg.
  • Rag2 rat recombinant activating gene 2
  • IL2rg interleukin 2 receptor ⁇
  • the method further comprises: disrupting the rat fumarate acetoacetate hydrolase (Fah) gene.
  • the second exon of the fumarate acetylacetate hydrolase gene is disrupted.
  • the third exon of recombinant activation gene 2 is disrupted.
  • the second exon of the interleukin 2 receptor gamma gene is disrupted.
  • the disruption is performed using the Crispr/Cas9 gene editing method.
  • the second exon of the fumarate acetoacetate hydrolase gene is disrupted by using an sgRNA targeting the second exon of the fumarate acetylacetate hydrolase gene and Cas9 mRNA; preferably, The sgRNAs are sgRNAs of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the third exon of recombinant activator gene 2 is disrupted by using sgRNA targeting the third exon of recombinant activator 2 and Cas9 mRNA; preferably, said sgRNA is SEQ ID NO: 3 and sgRNA of the nucleotide sequence shown by SEQ ID NO: 4.
  • the second exon of the interleukin 2 receptor ⁇ gene is disrupted by using an sgRNA targeting the second exon of the interleukin 2 receptor ⁇ gene and Cas9 mRNA; preferably, the The sgRNA is an sgRNA of the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.
  • the sgRNA and Cas9 mRNA or a construct capable of forming the sgRNA and Cas9 mRNA are introduced into a fertilized egg of a rat; the fertilized egg is developed to obtain an immunodeficient rat.
  • the method of preparing an immunodeficient rat or the method of preparing a rat cell is a "non-therapeutic purpose" method.
  • an sgRNA for use in the preparation of an immunodeficient rat which is directed to a third exon of recombinant activator gene 2, which is set forth in SEQ ID NO: 3 and SEQ ID NO: a sgRNA of a nucleotide sequence; and a second exon thereof which is targeted to the interleukin 2 receptor gamma gene, which is the sgRNA of the nucleotide sequence shown by SEQ ID NO: 5 and SEQ ID NO: 6.
  • an sgRNA targeting the second exon of the fumarate acetylacetate hydrolase gene which is the sgRNA of the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
  • kits for the preparation of an immunodeficient rat comprising: a sgRNA of the nucleotide sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 4; and/or
  • sgRNA of the nucleotide sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6.
  • the sgRNA further comprises: the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the kit further comprises Cas9 mRNA or a construct capable of forming Cas9 mRNA.
  • a method of preparing a rat transplantation model comprising:
  • the heterologous cells include, but are not limited to, hepatocytes, tumor cells, stem cells (eg, embryonic stem cells, hematopoietic stem cells), iPS cells, blood cells (eg, red blood cells, white blood cells).
  • stem cells eg, embryonic stem cells, hematopoietic stem cells
  • iPS cells eg, iPS cells
  • blood cells eg, red blood cells, white blood cells.
  • the heterologous cells are of human origin or of a mammal (e.g., a primate such as a monkey, a donkey, a rodent such as a mouse, etc.).
  • a mammal e.g., a primate such as a monkey, a donkey, a rodent such as a mouse, etc.
  • the immunodeficient rat is a rat having a recombinant activating gene 2 and an interleukin 2 receptor ⁇ gene deletion, and heterologous hepatocytes, tumor cells, stem cells (eg, embryonic stem cells, hematopoietic stem cells) ), iPS cells, blood cells (such as red blood cells, white blood cells) were transplanted into the rat to obtain a xenograft model.
  • the immunodeficient rat is a rat of the fumarate acetylacetate hydrolase gene, the recombinant activator gene 2, and the interleukin 2 receptor gamma gene deletion, and the heterologous hepatocytes, tumor cells, Stem cells (such as embryonic stem cells, hematopoietic stem cells), iPS cells, and blood cells (such as red blood cells, white blood cells) are transplanted into the rat to obtain a xenograft model.
  • Stem cells such as embryonic stem cells, hematopoietic stem cells
  • iPS cells iPS cells
  • blood cells such as red blood cells, white blood cells
  • liver diseases include, for example but are not limited to: hepatitis virus infection diseases, malaria.
  • FIG. 1 Construction of FRG rats using the CRISPR system.
  • FIG. 1 Schematic diagram of A, Fah, Rag2, IL2rg genes.
  • the amplified partial sequence is the sgRNA target.
  • F and R represent primers for genotypic identification.
  • Fah -/- rats can be used as a hepatocyte transplantation model.
  • Fah +/- rats were mated to obtain homozygotes.
  • NTBC is added to the drinking water during the pregnancy of the mother and after the birth of the mouse.
  • Fah - / - rats received liver cell transplantation (Fah - / - w / HTx) from the same nest wild type rats, NTBC water was removed immediately after transplantation. Kaplan-Meier survival curves of transplanted and non-transplanted hepatocyte Fah -/- rats.
  • Figure 3 Defects in thymus and spleen development of Rag2 -/- IL2rg -/- rat.
  • the 8-week-old Rag2 -/- , IL2rg -/- and Rag2 -/- IL2rg -/- rat serum immunoglobulins were quantitatively analyzed by ELISA.
  • A, B, C, 5 ⁇ 10 6 Snu-398 was transplanted into Rag2 -/- IL2rg -/- rat in the inguinal formation of subcutaneous tumor morphology (A), size (B) and H&E staining (C).
  • Fah -/- Rag2 -/- IL2rg -/- rats can be used as a model for efficient hepatocyte repopulation.
  • A, 3 ⁇ 10 6 mouse hepatocytes were transplanted into FRG rats. Rats were treated at 4 and 7 weeks, and the proportion of liver repopulation was determined by FAH staining.
  • liver sections of humanized rat liver were subjected to immunofluorescence staining of human-specific ALB, AAT, FAH, CYP3A4 and GS.
  • Quantitative PCR was used to detect the expression of important metabolic enzymes in the liver of humanized mouse liver tissue, primary human liver cells and liver humanized rat liver tissues.
  • the inventors After intensive research and experiments, the inventors first constructed Fah -/- Rag2 -/- IL2rg -/- immunodeficient rats by rational design based on Crispr/Cas9 gene editing technology. Further, the present inventors further applied the immunodeficient rat to establish a cell transplantation model such as hepatocytes and tumor cells.
  • the immunodeficient rat of the invention has a good application prospect.
  • the "sgRNA” is either “Single-guide RNA (sgRNA)” or “single-directed RNA", which is based on a "target site on a target gene” design that contains sufficient sequence Synergistic with endonuclease Cas9 leads to the occurrence of Cas9-mediated DNA double-strand breaks at the target site.
  • sgRNA Single-guide RNA
  • single-directed RNA which is based on a "target site on a target gene” design that contains sufficient sequence Synergistic with endonuclease Cas9 leads to the occurrence of Cas9-mediated DNA double-strand breaks at the target site.
  • the mammal refers to a mammalian animal, including humans, non-human primates (monkeys, orangutans), livestock and farm animals (eg, pigs, sheep, cattle), rats (mouse), and Rodents (eg, mice, rats, rabbits) and the like.
  • non-human primates monkeys, orangutans
  • livestock and farm animals eg, pigs, sheep, cattle
  • rats mouse
  • Rodents eg, mice, rats, rabbits
  • exogenous or heterologous refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between cells and hosts from different sources. For example, if the combination of a nucleic acid and a host cell is generally not naturally occurring, the nucleic acid is heterologous to the host cell. A particular sequence is “heterologous” to the cell or organism into which it is inserted.
  • introducing or “transforming” refers to the transfer of an exogenous polynucleotide into a host (in the present invention, an animal or animal cell).
  • construct includes “plasmid.”
  • the present invention provides a method for preparing an immunodeficient rat, comprising: disrupting a rat fumarate acetylacetate hydrolase (Fah) gene, a recombinant activating gene 2 (Rag2) and an interleukin 2 receptor ⁇ (IL2rg) gene; Obtained immunodeficient rats.
  • the disruption is performed using the Crispr/Cas9 gene editing method.
  • Fah is a 419 amino acid enzyme with a gene length of 22586 bp, 14 exons and 13 introns; its GenBank accession number is NM_017181.2.
  • the optimal position of the sgRNA design should be followed by the transcription start site ATG, such that the mutation causes the translation to terminate prematurely, forming a shorter non-functional truncated protein.
  • Fah's transcriptional transcription initiation site ATG is in the first exon, the inventors found that it is not suitable for sgRNA design. After repeated studies, analysis and experiments, the inventors determined that two sgRNAs were designed with the appropriate position of the second exon next to ATG as a target site.
  • the nucleotide sequence of the sgRNA is set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
  • Rag2 is a protein of 527 amino acids with a gene length of 8297 bp, with 3 exons and 2 introns; its GenBank accession number is NM_001100528.1.
  • the present inventors have designed, after repeated studies, analysis and experiments, to determine the appropriate position of the third exon in which the Rag2 transcription initiation site ATG is located as a targeting site, and design two sgRNAs.
  • the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3 and SEQ ID NO: 4.
  • IL2rg is a 368 amino acid protein with a gene length of 7281 bp, 12 exons and 11 introns; its GenBank accession number is NM_080889.1.
  • the present inventors have designed, after repeated studies, analysis and experiments, to design the two sgRNAs by using the appropriate position of the second exon in which the IL2rg transcription initiation site ATG is located as a targeting site.
  • the nucleotide sequence of the sgRNA is set forth in SEQ ID NO: 5 and SEQ ID NO: 6.
  • Fah gene knockout can cause inducible liver damage in rats.
  • Rag2 gene knockout can lead to the loss of T and B cells in rats.
  • IL2rg gene knockout can lead to the loss of NK cells and the decrease of T and B cells in rats.
  • Both Rag2 and IL2rg can be knocked out and knocked out to obtain immunodeficient rats, while Rag2 and IL2rg are knocked out at the same time, and the immunodeficiency is the most serious, suitable for human liver cell transplantation.
  • the immunodeficient rat can be conveniently prepared based on the Crispr/Cas9 gene editing method, and the step is simpler and more operable than the preparation of the transgenic rat.
  • the sgRNA can be synthesized by conventional techniques in the art, and then introduced into Cas9 mRNA into animal germ cells such as fertilized eggs, and Cas9 mRNA is translated into the active Cas9 enzyme in vivo, and the sgRNA is targeted in the genome.
  • the specific position guides the specific cleavage of the Cas9 enzyme, resulting in deletion and mutation of the specific position of the gene.
  • the fertilized egg is obtained from an immunodeficient rat after development.
  • the immunodeficient rat obtained by designing the selected sgRNA using the method of the present invention may be a Rat having a Rag2 gene and an IL2rg deletion; or a rat in which the Fah, Rag2 and IL2rg genes are deleted in common.
  • the immunodeficient rat obtained by the method of the present invention can be applied to prepare a rat transplantation model, or to amplify a heterologous cell (such as a liver cell, particularly a human liver cell), or to a drug research.
  • a heterologous cell such as a liver cell, particularly a human liver cell
  • a heterologous cell is transplanted in the immunodeficient rat of the present invention, thereby preparing a rat transplantation model.
  • the heterologous cells include, but are not limited to, non-rat-derived hepatocytes, tumor cells, stem cells (e.g., embryonic stem cells, hematopoietic stem cells), iPS cells, and blood cells (erythrocytes and white blood cells).
  • the heterologous cells may be of human origin or of a mammal (e.g., a primate such as a monkey, a donkey, a rodent such as a mouse, etc.).
  • a Fah -/- rat is obtained, which can be used as an animal model for liver cell transplantation and efficient reconstruction of the liver, and the wild-type rat liver cells can be transplanted into the animal model to obtain a high
  • the proportion of hepatocyte repopulation is higher than 90%.
  • the immunodeficient rat is a rat having a Rag2 gene and an IL2rg gene deletion (Rag2 -/- IL2rg -/- ), and heterologous hepatocytes, tumor cells, and stem cells (
  • a xenograft model can be obtained by transplanting the rat, such as embryonic stem cells, hematopoietic stem cells, iPS cells, blood cells (erythrocytes and white blood cells).
  • the immunodeficient rat is a Fah gene, a Rag2 and an IL2rg gene-deleted rat (Fah -/- Rag2 -/- IL2rg -/- ), and the heterologous liver Cells, tumor cells, stem cells (such as embryonic stem cells, hematopoietic stem cells), iPS cells, and blood cells (erythrocytes and white blood cells) were transplanted into the rat to obtain a xenograft model.
  • the repopulation rate of human hepatocytes in the liver of Fah -/- Rag2 -/- IL2rg -/- rats was as high as 65%.
  • Rats transplanted with human hepatocytes were used as bioreactors to produce human hepatocytes.
  • Human hepatocytes are grown in rats to obtain humanized liver animals for scientific research as well as clinical.
  • the rats can be used as a transplant tumor model for scientific research and drug screening, drug metabolism and toxicological testing.
  • Hematopoietic stem cells and blood cells are transplanted, and rats with humanized blood system can be obtained for scientific research and drug screening.
  • the immunodeficient rat of the present invention has the advantages of inducing liver injury characteristics, high hepatocyte repopulation ratio, high degree of immunodeficiency, and relatively easy feeding.
  • human hepatocytes it is difficult for human hepatocytes to be expanded in large amounts under ex vivo conditions, and in the immunodeficient rats of the present invention, a large amount of amplification can be performed, and the obtained human hepatocytes are not only metabolically active but also freshly isolated human hepatocytes. Consistently, more liver-humanized rats can be transplanted. Compared with hepatocytes isolated from human liver, these human hepatocytes isolated from liver-humanized rats are more likely to be frozen and have a better adherence effect after resuscitation. Therefore, humanized rat liver can be used as a bioreactor for large-scale production of human hepatocytes for high-throughput drug screening, toxicological analysis, and bioartificial liver.
  • the invention also provides a kit for preparing an immunodeficient rat, the kit comprising sgRNA and Cas9 mRNA for performing the CRISPR/Cas9 method operation or a reagent capable of forming the sgRNA and Cas9 mRNA in vivo or in vitro .
  • kits to facilitate use by those skilled in the art, such as microinjection reagents and the like.
  • instructions for use by those skilled in the art may also be included in the kit.
  • Rat primary hepatocytes were freshly isolated from wild-type SD rats, and primary mouse hepatocytes were isolated from wild-type C57 mice, and stored at 4 ° C or on ice, and transplanted within 6 hours.
  • Frozen human liver cells were purchased from Shanghai Ruide Liver Company.
  • Rabbit anti-Fah (AbboMax, 1:3000), Rabbit anti-Ki67 (Novocastra, 1:1000, antigen-repairing), Goat anti-human ALB (Bethyl, 1:200), Rabbit anti-CYP3A4 (Santa Cruz, 1 : 200, antigen retrieval required), Rabbit anti-AAT (NeoMarkers, 1:200), Mouse anti-GS (BD, 1:100). .
  • Rat immunoglobulin was quantified using Rat IgG, IgA, IgM Ready-SET-Go! ELISA kit (eBioscience).
  • the KOD enzyme used to construct the cloned DNA was purchased from Toyobo Corporation; DNA sequencing was performed by Huada Gene or Shanghai Yingjie Jieji Company.
  • 10 ⁇ EBSS buffer (excluding Ca 2+ and Mg 2+ ): 65.7 g NaCl, 2.91 g Na 3 PO 4 ⁇ 12H 2 O, 3.9 g KCl, 9.7 g D-glucose dissolved in 1 L of deionized water, adjusted The pH is up to 7.4.
  • 10 ⁇ EBSS buffer (containing Ca 2+ and Mg 2+ ): 1.325 g CaCl 2 , 2 g KCl, 0.985 g MgSO 4 , 34 g NaCl, 11 g NaHCO 3 , 0.78 g NaH 2 PO 4 ⁇ H 2 O, 5 g D-glucose is dissolved in 500 ml of deionized water and does not require adjustment of the pH.
  • Liver perfusion solution I 10 ml of 10 ⁇ EBSS buffer (without Ca 2+ and Mg 2+ ), 0.5 ml of 100 mM EGTA, deionized water to 100 ml, and adjusted to pH 7.4.
  • Liver perfusion solution II 10 ml of 10 ⁇ EBSS buffer (containing Ca 2+ and Mg 2+ ), 1 ml of 1 M Hepes was added with deionized water to 100 ml, and the pH was adjusted to 7.4.
  • Liver perfusion buffer III 20 ml of 10 ⁇ EBSS buffer (containing Ca 2+ and Mg 2+ ), 2 ml of 1 M Hepes, 50 mg of type IV collagenase, deionized water to 200 ml, and adjusted to pH 7.4.
  • Fah -/- , Rag2 -/- IL2rg -/- and Fah -/- Rag2 -/- IL2rg -/- (FRG) rats are SD background, and Fah -/- and FRG rats need to be fed with 5.4mg /L NTBC water. All animals were cultured in an SPF animal house. Animal experiments were carried out in strict accordance with the animal platform operating regulations and animal welfare.
  • the primers used to detect the Fah, Rag2, IL2rg genotypes in the examples are shown in Table 1.
  • Rats (5-8 weeks, about 200 g) were anesthetized and intraperitoneally injected with 6 mg/ml sodium phenobarbital (5-10 ul/g body weight).
  • Solution I was perfused with 50 to 100 ml, and the liver was changed to blood after coloring.
  • Solution II is perfused with 50-100 ml, and then changed to solution III (the peristaltic pump should be suspended when changing the fluid).
  • Solution III is perfused with 400 ml until the liver tissue has a certain fluidity, and the epidermis is separated from the liver parenchyma.
  • Hepatocytes were resuspended in 10 ml DMEM medium, counted and viable.
  • Rats (5-8 weeks, about 200 g) were anesthetized and intraperitoneally injected with 6 mg/ml phenobarbital (5-10 ul/g body weight).
  • the rat liver was fixed overnight with 4% PFA, dehydrated and embedded, and cut into 3 um thick.
  • the capture antibody was diluted 1:250 with a coating buffer, and 100 ul per well was added to a 96-well ELISA plate. The 96-well plates were sealed and incubated overnight at 4 °C.
  • the rat spleen was placed in a 10 cm dish containing 2% FBS DMEM.
  • the culture dish should be placed on ice.
  • the snu398 cell line obtained from ATCC was digested, and 5 ⁇ 10 6 cells were directly injected into the groin of immunodeficient rats.
  • tumor volume a x b 2 /2, a for the longest side and b for the shortest side.
  • the tumor was fixedly embedded with 4% PFA and histologically tested.
  • RNA extracted by Trizol 1 ug or whole of RNA extracted by RNeasy FFPE Kit, and cDNA obtained by M-MLV reverse transcriptase (Promega) kit.
  • sgRNA-1 CCACGGATTGGTGTGGCCATCGG (SEQ ID NO: 1);
  • sgRNA-2 ATCGAAGACATGCTGATGTTTGG (SEQ ID NO: 2);
  • sgRNA-1 CCTAAGAGATCCTGCCCTACTGG (SEQ ID NO: 3);
  • sgRNA-2 ACGAAGAGGTGGGAGGTAGCAGG (SEQ ID NO: 4);
  • sgRNA-1 AGGAGTAAGAAGGATCTAGATGG (SEQ ID NO: 5);
  • sgRNA-2 TCCAAGGTCCTCATGTCCAGTGG (SEQ ID NO: 6).
  • Cas9 mRNA and 6 sgRNAs were microinjected into the fertilized eggs of SD rats and then transferred to the uterus of pseudopregnant mothers.
  • Genomic DNA was extracted from the tail of neonatal rat and PCR amplified using a pair of primers at both ends of the Fah, Rag2, and IL2rg genes (Fig. 1A).
  • Fig. 1B By DNA sequencing, the inventors identified a newborn mouse with a mutation of three genes (Fig. 1B).
  • the present inventors obtained the F1 generation rat by breeding the newborn rat with the mutation of the three genes obtained above as a Founder rat and a wild type rat. PCR genotype identification of F1 rats also revealed deletion mutations with these three genes, indicating that these mutations are stably inherited (Fig. 1C).
  • Fah -/- , Rag2 -/- ILrg -/- and Fah -/- Rag2 -/- IL2rg -/- (FRG) rats were obtained by selfing the F1 generation rats.
  • Fah -/- rats can be used as an animal model for hepatocyte transplantation and efficient reconstruction of the liver.
  • Fah -/- rats were first obtained. Fah -/- rats were able to grow normally with NTBC feeding, and Fah expression was completely absent in the liver (Fig. 2A and B). When NTBC was withdrawn, Fah -/- rats continued to lose weight and eventually died of liver failure ( Figures 2C and D). After the spleen transplantation of wild-type rat hepatocytes, the body weight of Fah -/- rats gradually recovered after 2 weeks of decline, and about 60% of the rats could eventually survive (Fig. 2C and D). By Fah immunohistochemical staining, it was found that Fah-positive hepatocytes were repopulated in large numbers, and the repopulation ratio was >90%, and the entire rat liver was almost reconstituted (Fig. 2E).
  • the inventors After selfing of the F1 generation, the inventors obtained Rag2 -/- IL2rg -/- rats (Fig. 3A). It can grow normally in the SPF environment, and it is no different from wild-type rats and can survive for more than 1.5 years. The thymus and spleen are the two most important lymphoid organs in animals. By dissecting a 5-week-old rat at the developmental stage, the inventors found that wild-type rats have distinct thymus tissue, while Rag2 -/- , IL2rg -/-, and Rag2 -/- IL2rg -/- The thymus develops severely in the mouse (Fig. 3B).
  • HE staining further confirmed that Rag2 -/- , IL2rg -/- and Rag2 -/- IL2rg -/- rats had only epithelioid cells and no lymphocytes relative to the wild type rat thymus (Fig. 3C).
  • the white pulp in the spleen contains B cells and T cells, and the red pulp contains more red blood cells.
  • CD3 - CD45RA + B cells and CD3 + CD45RA - T cells completely disappeared in the spleen of Rag2 -/- rats, but CD161a + NK cells were present and significantly increased (Fig. 5D and E).
  • CD3 - CD45RA + B cells and CD3 + CD45RA - T cells were significantly decreased in IL2rg -/- rats compared to wild-type animals, while CD161a + NK cells also disappeared (Fig. 5D and E). This indicates that IL2rg knockdown not only affects NK cell development, but also affects T and B cell development. T, B and NK cells were almost completely absent in Rag2 -/- IL2rg -/- rats (Fig. 5D and E).
  • Immunodeficient mice are widely used for xenografts, particularly human tumor cells, embryonic stem cells, hematopoietic stem cells, and hepatocytes.
  • Immunodeficient mice are also commonly used in teratoma formation experiments to detect pluripotency of iPS cells.
  • mice hepatocytes After obtaining FRG rats by breeding, the inventors first transplanted 3 ⁇ 10 6 mouse hepatocytes for detecting whether or not they can receive heterologous hepatocytes. Rats were treated at 4 and 7 weeks of transplantation and immunohistochemical staining with Fah showed that mouse hepatocytes were extensively repopulated in the liver of FRG rats, and the repopulation ratio was 86% (Fig. 7A). It is indicated that heterologous hepatocytes can also completely reconstitute the liver of FRG rats.
  • the present invention further transplanted with 2x10 6 cryopreserved primary human hepatocytes into rat FRG.
  • a large amount of human albumin secretion (112-560 ug/ml) was found in the serum.
  • the amount of albumin secretion was found to increase to 1.6 mg/ml.
  • Fah immunohistochemical staining revealed that Fah-positive human hepatocytes were integrated in the liver of FRG rats (Fig. 7B) with an integration ratio of up to 25%.
  • Fah -/- Rag2 -/- IL2rg -/- rats can be used to amplify human hepatocytes in vivo and construct liver-humanized rats.
  • the present invention is transplanted 2 ⁇ 10 6 cryopreserved primary hepatocytes FRG to rat liver and obtained a high proportion of humanized rat FRG 5 months after transplantation.
  • human-specific ALB and AAT staining it was found that the proportion of human hepatocytes repopulated in the liver of FRG rats reached 46 ⁇ 15% (Fig. 8A).
  • Human liver metabolic enzymes have the characteristics of regional expression, such as expression of liver phase I metabolic enzymes, glutamine synthetase (GS) in the central vein, and expression of urea metabolism-related enzymes in the portal vein.
  • the present inventors also found in human livers of a high proportion of liver-derived rats that human liver cells not only express the liver marker genes ALB, AAT and FAH, but also specifically express CYP3A4 and GS in the central vein (Fig. 8B).
  • liver-derived rats expressed liver phase I metabolic enzymes, including CYP3A4, CYP2A6, CYP2C9, CYP2E1, etc., phase II metabolic enzymes such as GSTA1, UGT2B7, etc., transporters such as SLC2A2, SLC22A1 , SLCO1B1, etc. (Fig. 8C), and expression levels are comparable to primary human hepatocytes and liver humanized mice.
  • phase I metabolic enzymes including CYP3A4, CYP2A6, CYP2C9, CYP2E1, etc.
  • phase II metabolic enzymes such as GSTA1, UGT2B7, etc.
  • transporters such as SLC2A2, SLC22A1 , SLCO1B1, etc.
  • liver-derived FRG rats have established human liver-specific metabolic gene expression and structure, which can be used for liver metabolism research and drug development experiments.

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Abstract

La présente invention concerne un procédé de préparation de rats immunodéficients et son application. De nouveaux rats immunodéficients sont obtenus au moyen d'un nouveau procédé. Au moyen des rats immunodéficients, on établit ensuite un modèle de transplantation de cellules telles que des cellules hépatiques et des cellules tumorales. Les rats immunodéficients ont une bonne perspective d'application.
PCT/CN2018/091503 2017-06-16 2018-06-15 Procédé de préparation de rats immunodéficients et utilisation associée WO2018228534A1 (fr)

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CN112616775B (zh) * 2020-12-30 2023-02-10 昕慕（上海）科技发展有限公司 一种采用复合因子法建立健康sd雄性大鼠肝纤维化方法

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