WO2018228534A1 - Method for preparing immunodeficient rats and application thereof - Google Patents

Abstract

A method for preparing immunodeficient rats and an application thereof. Novel immunodeficient rats are obtained by a new method. By applying the immunodeficient rats, a model for cell transplantation such as hepatic cells and tumor cells is further established. The immunodeficient rats have a good application prospect.

Description

Method for preparing immunodeficient rats and application thereof Technical field

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.

Background technique

Liver disease is one of the most important diseases that threaten human health. In particular, 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. However, 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. Previously, HBV drug non-auritoin failed in clinical stage II due to severe liver toxicity resulting in 5 deaths. However, it was not found to have hepatotoxicity in dogs, monkeys, rats, and mice. 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.

In 1990, Brinster Laboratories constructed 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. By inhibiting the complement response and selecting proliferative human hepatocytes, Katsutoshi Yoshizato Laboratories achieved a very high proportion (>90%) of human hepatocyte repopulation in uPA/SCID mice. In this highly repopulated liver humanized mouse, the expression and induction of human metabolic enzymes are very close to human liver. However, 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 (normal expression of FAH gene) can almost completely reconstitute Fah ^-/- mice after spleen transplantation. The liver (over 90%), the recipient mice returned to normal liver function. After 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. Based on Nod/SCID/IL2rg ^-/- mice (NOG mice), the Herpes simplex virus type 1thymidine kinase was specifically expressed in the liver using the Alb promoter. By injecting GCV into mice, 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. However, this immunodeficient mouse can only be propagated by artificial insemination, and the feeding environment is extremely clean and difficult to promote.

Su Lishan Laboratories established a dual-source mouse model of liver and immune system to specifically express FK506-binding protein (FKBP) in the liver using the Alb promoter on the basis of Rab2 ^-/- IL2rg ^-/- mice in the Balb/c background. And a fusion protein of caspase 8 (Caspase 8). Induction of FKBP to form a dimer by injection of AP20187 to activate Caspase8 leads to apoptosis of mouse hepatocytes, and at the same time promotes repopulation of human hepatocytes. 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. However, 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. In addition, the relatively low proportion of human hepatocyte repopulation (10-30%) and the relative immatureness of hepatic progenitors are defects in this model.

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. In theory, 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.

In recent years, various immunodeficient rats such as Rag1/2 ^-/- , IL2rg ^-/- and Prkdc ^-/- IL2rg ^-/- have been reported. However, these immunodeficient rats are mainly constructed by using Zinc-finger nuclease and TALEN technology, which are costly and complicated, and they have problems of reproductive difficulty, especially the premature aging of Prkdc ^-/- IL2rg ^-/- rats. This is not conducive to promotion and industrial use. Currently, in Rag1 ^-/- immunodeficient rats, human primary hepatocytes can achieve a 10% repopulation ratio in rat liver. This suggests that similar to mice, the construction of liver-humanized rats is also possible. However, 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. Especially for female animals, even if the time after transplantation is extended to 1 year, only 40-60% of the repopulation ratio can be achieved, and the humanized rat liver with high human liver cell repopulation rate has not been successfully obtained. Therefore, 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.

Summary of the invention

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.

In a first aspect of the invention, a method for producing an immunodeficient rat comprising: disrupting a rat recombinant activating gene 2 (Rag2) and an interleukin 2 receptor γ (IL2rg) gene; thereby obtaining an immunodeficient rat .

In another aspect of the present invention, a method for producing a rat cell, comprising: disrupting a rat recombinant activating gene 2 (Rag2) and an interleukin 2 receptor γ (IL2rg) gene; preferably, the cell is Fertilized egg.

In a preferred embodiment, the method further comprises: disrupting the rat fumarate acetoacetate hydrolase (Fah) gene.

In another preferred embodiment, the second exon of the fumarate acetylacetate hydrolase gene is disrupted.

In another preferred embodiment, the third exon of recombinant activation gene 2 is disrupted.

In another preferred embodiment, the second exon of the interleukin 2 receptor gamma gene is disrupted.

In another preferred embodiment, the disruption is performed using the Crispr/Cas9 gene editing method.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the second exon of the interleukin 2 receptor γ gene is disrupted by using an sgRNA targeting the second exon of the interleukin 2 receptor γ 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.

In another preferred embodiment, 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.

In another preferred embodiment, the method of preparing an immunodeficient rat or the method of preparing a rat cell is a "non-therapeutic purpose" method.

In another aspect of the invention, there is provided 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.

In a preferred embodiment, further comprising: 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.

In another aspect of the invention, there is provided a kit 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.

In a preferred embodiment, the sgRNA further comprises: the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

In another preferred embodiment, the kit further comprises Cas9 mRNA or a construct capable of forming Cas9 mRNA.

In another aspect of the invention, a method of preparing a rat transplantation model is provided, comprising:

(1) preparing an immunodeficient rat by the method described in any of the preceding;

(2) In the immunodeficient rats of (1), heterologous cells were transplanted, and a rat transplantation model was obtained.

In a preferred embodiment, 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).

In another preferred embodiment, 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.).

In another preferred embodiment, the immunodeficient rat is a rat having a recombinant activating gene 2 and an interleukin 2 receptor γ 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.

In another preferred embodiment, 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.

In another aspect of the invention, there is provided the use of a rat transplantation model obtained by the method described above for: drug metabolism and toxicology detection; liver disease and tumor research and drug screening; or as a bioreactor producer Hepatocyte. The liver diseases include, for example but are not limited to: hepatitis virus infection diseases, malaria.

Other aspects of the invention will be apparent to those skilled in the art from this disclosure.

DRAWINGS

Figure 1. Construction of FRG rats using the CRISPR system.

Schematic diagram of A, Fah, Rag2, IL2rg genes. The amplified partial sequence is the sgRNA target. F and R represent primers for genotypic identification.

B. DNA sequencing results and protein sequence prediction results of Fah, Rag2, IL2rg genes in the Founder rat with mutation. The dotted line indicates the deleted base and the inside of the box is the added base.

C, Founder rats and WT rats were backcrossed to obtain F1 rat gene identification results.

Figure 2. Fah ^-/- rats can be used as a hepatocyte transplantation model.

Photographs of Fah ^-/- and WT rats at A and 4 weeks. 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 immunohistochemical staining of B, 4 week old WT and Fah ^-/- rat fed NTBC.

C, 6-8 week old 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.

D. Changes in body weight of transplanted and non-transplanted hepatic Fah ^-/- rats. The body weight was weighed once a week, and all values were relative values calculated on day 0 of body weight 1.

E, 10 ⁷ Wild-type hepatocytes from littermates were transplanted into Fah ^-/- rats. Liver repopulation was identified by Fah immunohistochemical staining at 4 and 8 weeks after transplantation, and the proportion of repopulation was quantified.

Figure 3. Defects in thymus and spleen development of Rag2 ^-/- IL2rg ^-/- rat.

A, Rag2 ^-/- and Rag2 ^-/- IL2rg ^-/- rat photo and genotype identification results.

B, 5 weeks old Rag2 ^{- / -} , IL2rg ^{- / -} and Rag2 ^{- / -} IL2rg ^{- / -} rat thymus.

H and E staining results of thymus and spleen of C, 5 week old Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- rats.

Figure 4. Rat immunoglobulin secretion by Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- .

The 8-week-old Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- rat serum immunoglobulins were quantitatively analyzed by ELISA.

Figure 5. Development of immune cells in thymus and spleen of Rag2 ^-/- IL2rg ^-/- rat

A, B, C, Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- CD4 ⁺ (A) or CD8 ⁺ (B) single positive T cells and CD4 ⁺ CD8 ⁺ double yang in the thymus of rats Development of sex T cells (C).

D, E, Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- in the spleen of rats, CD3 ^- CD45RA ⁺ B cells, CD3 ⁺ CD45RA ^- T cells (D) and CD161a ⁺ NK cells (E ) development.

Figure 6. Rag2 ^-/- IL2rg ^-/- rats can be used as a xenograft model.

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).

D, 5 × 10 ⁶ human iPS cells were transplanted into Rag2 ^{- / -} IL2rg ^{- / -} rat testicular cysts to form teratoma H & E staining.

Figure 7. 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.

B. Fah immunohistochemical staining of FRG rats (FRG W/HTx) liver with human albumin secretion after transplantation of human liver cells.

C. After continuous sectioning of the liver, specific recognition of human albumin (ALB) and Ki67 immunohistochemical staining was performed.

D. Immunohistochemical staining of FAH and CYP3A4 was performed after serial sectioning of the liver.

Figure 8. High proportion of liver humanized FRG rats have human liver specific metabolic gene expression and structure.

A. After transplanting human liver cells for 5 months, the rat liver was subjected to immunofluorescence staining of human-specific ALB and AAT. The proportion of human hepatocyte integration was statistically based on staining positive immunization.

B. Liver sections of humanized rat liver were subjected to immunofluorescence staining of human-specific ALB, AAT, FAH, CYP3A4 and GS.

C. 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.

detailed description

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.

As used herein, 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.

As used herein, 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.

As used herein, "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.

As used herein, the term "introducing" or "transforming" refers to the transfer of an exogenous polynucleotide into a host (in the present invention, an animal or animal cell).

As used herein, "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 γ (IL2rg) gene; Obtained immunodeficient rats. Preferably, 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. Although 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. Thus, as a preferred mode of the invention, 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. As a preferred mode of the present invention, 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. Thus, as a preferred mode of the invention, 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.

A variety of methods known in the art for disrupting genes can be used to disrupt the gene under the direction of the present invention. Preferably, 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.

As an application, 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.).

As an embodiment of the present invention, 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%.

As an embodiment of the present invention, 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).

As another embodiment of the present invention, 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.

Rats transplanted with tumor cells, tumor cells grow in rats and form tumors. 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.

Compared with other animal models, 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.

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 .

Other reagents commonly used for transgenic manipulations can also be included in the kits to facilitate use by those skilled in the art, such as microinjection reagents and the like. In addition, instructions for use by those skilled in the art may also be included in the kit.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Materials and Method

cell

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.

antibody

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). .

Reagent

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.

Liver perfusion involves buffer:

1) 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.

2) 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.

3) 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.

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.

5) 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.

Experimental animal

Wild type SD rats and wild type C57 mice, 6-8 weeks, were purchased from Shanghai Slack Company.

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.

Primer information

The primers used to detect the Fah, Rag2, IL2rg genotypes in the examples are shown in Table 1.

Table 1

Microinjection of sgRNA and Cas9 mRNA

1) The super-treated SD mother rats and male rats were mated to obtain fertilized eggs, which were placed in KSOM embryo culture medium.

2) A TE solution containing 12.5 ng/ul sgRNA and 25 ng/ul Cas9 mRNA was injected into a cell-stage embryo using a microinjection needle.

3) The fertilized egg after microinjection is transplanted into the uterus of the pseudopregnant female. Gene knockout newborn mice were obtained 21 days later.

Rat primary hepatocyte isolation

1) Preheat the three perfusate in a 37 ° C water bath.

2) Fix the infusion tube in the peristaltic pump, one end is placed under the liquid surface of the solution I, the other end is connected with the needle, and the peristaltic pump is turned on to adjust the rotation speed to 11 to make the liquid fill the pipeline.

3) Rats (5-8 weeks, about 200 g) were anesthetized and intraperitoneally injected with 6 mg/ml sodium phenobarbital (5-10 ul/g body weight).

4) After the rats were anesthetized, the limbs were fixed on a foam pad, and a tray was placed under the foam pad. The ventral fur is cut longitudinally from the lower abdomen to the neck, and the fur is fixed to the left and right sides. Cut the abdominal wall laterally from the lower part of the abdomen, cut it along the left and right sides of the body to the lower part of the neck (cut the diaphragm and ribs in the middle), fix the abdominal wall to the right side of the head, fully expose the heart and liver, and dial with a cotton swab. Open abdominal fat and small intestine, expose the inferior vena cava and portal vein.

5) Insert the needle from the heart sinus into the superior vena cava (be careful not to puncture the superior vena cava), open the peristaltic pump power supply, you can see the liver, the inferior vena cava and the hepatic portal vein are filled and bulged, the hepatic portal vein is cut, and the hemostat is clamped. The inferior vena cava begins to perfusion.

6) 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).

7) Solution III is perfused with 400 ml until the liver tissue has a certain fluidity, and the epidermis is separated from the liver parenchyma.

8) Turn off the peristaltic pump, pull out the needle, cut the liver, place it in a 10cm dish, add 10ml of solution III, squeeze the liver with a blue gun head, squeeze out the liver cells, and discard the liver epidermis.

9) Add the hepatocyte suspension to 10 ml of medium, blow it to spread the cells, transfer to a centrifuge tube, 1000 RPM, 5 min, discard the supernatant.

10) 18 ml percol + 2 ml 10 × EBSS + 20 ml DMEM was mixed, and the precipitated liver cells were suspended, 1000 RPM, 5 min, and the supernatant was discarded.

11) Resuspend the cells with 2 ml ACK red blood cell lysate, glaze for 2-3 minutes, then neutralize with 10 ml DMEM medium, 1000 RPM, 5 min, discard the supernatant.

12) Hepatocytes were resuspended in 10 ml DMEM medium, counted and viable.

Rat primary hepatocytes and human primary hepatocyte transplantation

1) Rats (5-8 weeks, about 200 g) were anesthetized and intraperitoneally injected with 6 mg/ml phenobarbital (5-10 ul/g body weight).

2) The spleen side of the rat is facing upward, and the epidermis and muscle layer are cut at 1-2 cm below the rib. Find the brown fat attached to the spleen and pull it out to pull out the spleen. Gently ligature the anterior spleen with a thread.

3) Take 2-10×10 ⁶ rat hepatocytes or human primary hepatocytes (about 500 ul) with insulin, and insert the needle into the spleen beyond the ligation site. After the injection is complete, pause for 30-60 seconds, then pull out the needle and tighten the wire to prevent backflow of liquid.

4) Put the spleen into the abdominal cavity, suture the muscles and epidermis, disinfect the wound with alcohol cotton, and put it into the cage.

Immunohistochemistry

The rat liver was fixed overnight with 4% PFA, dehydrated and embedded, and cut into 3 um thick.

1) Dewaxed xylene 3 times for 5 minutes each time.

2) Gradient rehydration: 100% ethanol, 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, each for 5 minutes. Deionized water, soak for 1 minute.

3) Antigen repair (this step is optional according to antibody characteristics): Sodium citrate antigen repair solution with pH=6.0 is heated at 95 ° C for half an hour, and then naturally cooled to room temperature. Wash with deionized water for 15 minutes.

4) Soak in PBS for later use.

5) Circle the sample with ImmunoPen and drop a drop of 3% H ₂ O ₂ at room temperature for 15 minutes to block the endogenous peroxidase.

6) Wash twice with PBS for 5 minutes each time.

7) Block with 1:20 normal horse serum (dissolved in 1% BSA-PBS), incubate for 20 minutes at room temperature, and wash 3 times with PBS.

8) Add the primary antibody, place the slice in a wet box, seal it, and overnight at 4 °C.

9) Wash PBS three times for 5 minutes each time.

10) Add secondary antibody and incubate for 30 minutes at room temperature. Wash PBS three times for 5 minutes each time.

11) Add AB solution in ABC kit (diluted 1:100 in A and B) and incubate for 30 minutes at room temperature. Wash PBS three times for 5 minutes each time.

12) DAB color development, adjusting the color development time according to different antibodies.

13) Hematoxylin counterstained for 20 minutes. Wash with tap water for half an hour.

14) Gradient dehydration: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, each for 2 minutes.

15) After 15-30 minutes of xylene, the neutral resin is stored in a sheet.

Immunofluorescence

1) The dewaxing and rehydrating steps are the same as 1-3 in the immunohistochemical step.

2) Circle the sample with ImmunoPen, block with 1:20 Normal horse serum (dissolved in 3% BSA-PBS), incubate for 20 minutes at room temperature, and wash 3 times with PBS.

3) Add primary antibody (dissolved in 3% BSA-PBS), place the sections in a wet box, and seal at 4 degrees overnight.

4) Wash 3 times with PBS.

5) Add the corresponding fluorescent secondary antibody (dissolved in 3% BSA-PBS), incubate for 1 hour at room temperature, and wash 3 times with PBS.

6) Drop the fluorescent anti-quenching seal tablet.

Immunoglobulin ELISA

Use Rat IgG, IgA, IgM Ready-SET-Go! ELISA kit (eBioscience) kit. Proceed as follows:

1) 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.

2) After the capture antibody was aspirated, 400 ul per well of wash buffer was added, soaked for 1 minute, then aspirated and washed twice. The second time you need to wash the wash buffer.

3) Add 250 ul of blocking buffer and incubate for 2 hours at room temperature.

4) Wash in accordance with step 2, 2 times.

5) Dilute the standard twice with Assay buffer A and dilute 8 gradients. The sample is also estimated to be diluted according to the standard curve to ensure that the final OD450 falls within the calibration range.

6) 100 ul of standard and sample were added to a 96-well plate, blocked, and incubated for 2 hours at room temperature.

7) Wash according to step 2, 4 times.

8) Add 100 ul of detection antibody to each well and incubate for 1 hour at room temperature.

9) Wash according to step 2, 4 times.

10) Add 100 ul of substrate solution to each well and incubate for 15 minutes at room temperature.

11) Add 100 ul of stop solution to each well for A450 and A570 measurements. Subtract the A450 from the A570, which is the last reading for analysis.

Immune cell separation

For the thymus:

1) Take the rats within 4-8 weeks, open the chest, and attach the thymus to the surface of the heart. For immunodeficient animals, if the thymus is not obvious, the fat on the surface of the heart is removed and placed in a 10 cm dish containing 2% FBS DMEM. The culture dish should be placed on ice.

2) Tear the thymus with tweezers and tear it into small pieces as much as possible. During the tearing process, the cells are scattered in the medium.

3) The cell suspension was filtered through a 100 um filter and counted.

For the spleen:

1) The rat spleen was placed in a 10 cm dish containing 2% FBS DMEM. The culture dish should be placed on ice.

2) After cutting the spleen into small pieces, tear it with tweezers.

3) The cell suspension was filtered through a 100 um filter to remove tissue debris. Centrifuge at 1000 rpm for 5 minutes at 4 °C.

4) Resuspend in 1 ml Ack buffer, lyse on ice for 3 minutes, and neutralize by adding 6 ml of medium.

5) After centrifugation at 1000 rpm for 5 minutes at 4 ° C, it was resuspended in medium and counted.

For the bone marrow:

1) Peel the long bones of the hind legs of the rats and remove the ends with scissors.

2) Pipette 2% FBS DMEM with a 1 ml syringe, inject into the long bone section, and the solution flows out from the other end.

3) Collect cell suspension, filter through 100um filter to remove tissue debris. Centrifuge at 1000 rpm for 5 minutes at 4 °C.

4) Resuspend in 1 ml Ack buffer, lyse on ice for 3 minutes, and neutralize by adding 6 ml of medium.

5) After centrifugation at 1000 rpm for 5 minutes at 4 ° C, it was resuspended in medium and counted.

Immune cell flow analysis

1) Resuspend 5 x 10 ⁶ cells with 50-100 ul staining buffer (DMEM + 0.1% BSA). Add a straight-labeled primary antibody and each antibody requires an antibody-free control. Incubate at 4 ° C for 30-60 minutes.

2) 1 ml of the staining solution was added, washed once, and centrifuged at 1000 rpm for 5 minutes.

3) The supernatant was removed and resuspended in 200-300 ul PBS for flow cytometry. The voltage was adjusted using the antibody-free group as a negative control.

Subcutaneous tumor formation in liver cancer

1) The snu398 cell line (obtained from ATCC) was digested, and 5 × 10 ⁶ cells were directly injected into the groin of immunodeficient rats.

2) The rats were treated three weeks later and tumors were taken. The size of the tumor is calculated using the following formula: 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.

iPSC teratoma experiment

1) After digesting human iPSC, 5 × 10 ⁶ cells were resuspended in 300 ul of PBS and injected into the testes of immunodeficient rats.

2) After 8 weeks, the rat testes were fixed with 4% PFA and histologically examined.

HE staining

1) The dewaxing and rehydrating steps are the same as 1)-4) in the immunohistochemistry step.

2) Hematoxylin counterstained for 20 minutes. Wash with tap water for half an hour.

3) Gradient dehydration: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, each for 2 minutes.

4) After 15-30 minutes of xylene, the neutral resin is stored in a sheet.

Gene expression detection in humanized FRG rats

1) Liver-derived mouse and rat liver tissues and human primary hepatocytes were obtained, and cellular RNA was extracted using Trizol (Invitrogen) reagent.

2) 1 ug of 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.

3) Real-time quantitative PCR was performed using a SYBR Premix Ex Taq (TaKaRa) kit, and gene expression was detected using an ABI StepOne Plus real-time PCR system (Applied Biosystems). Primers are shown in Table 2.

Table 2

gene	Forward (5’-3’)	Reverse (5’-3’)
ALB	GCCTTTGCTCAGTATCTT (SEQ ID NO: 13)	AGGTTTGGGTTGTCATCT (SEQ ID NO: 14)
AAT	TATGATGAAGCGTTTAGGC (SEQ ID NO: 15)	CAGTAATGGACAGTTTGGGT (SEQ ID NO: 16)
CYP3A4	TTCAGCAAGAAGAACAAGGACAA (SEQ ID NO: 17)	GGTTGAAGAAGTCCTCCTAAGC (SEQ ID NO: 18)
CYP2A6	CAGCACTTCCTGAATGAG (SEQ ID NO: 19)	AGGTGACTGGGAGGACTTGAGGC (SEQ ID NO: 20)
CYP2E1	ATGTCTGCCCTCGGAGTCA (SEQ ID NO: 21)	CGATGATGGGAAGCGGGAAA (SEQ ID NO: 22)
FAH	CCTACGGCGTCTTCTCGAC (SEQ ID NO: 23)	CTGCAAGAACACTCTCGCCT (SEQ ID NO: 24)
GAPDH	CCACCTTTGACGCTGGG (SEQ ID NO: 25)	CATACCAGGAAATGAGCTTGACA (SEQ ID NO: 26)
UGT2B7	TCAGCCCTGGCCCAGATCCC (SEQ ID NO: 27)	ACAGCTGCTCCCCTGGCCTT (SEQ ID NO: 28)
Cyp2C9	GCCTGCCCCATGCAGTGACC (SEQ ID NO: 29)	CACAGCAGCCAGCCAGGCCAT (SEQ ID NO: 30)
CYP7A1	AGAAGCATTGACCCGATGGAT (SEQ ID NO: 31)	AGCGGTCTTTGAGTTAGAGGA (SEQ ID NO: 32)
HNF4A	CCACGGGCAAACACTACGG (SEQ ID NO: 33)	GGCAGGCTGCTGTCCTCAT (SEQ ID NO: 34)
GSTA1	CTGCCCGTATGTCCACCTG (SEQ ID NO: 35)	AGCTCCTCGACGTAGTAGAGA (SEQ ID NO: 36)
SLC2A2	GCTGCTCAACTAATCACCATGC (SEQ ID NO: 37)	TGGTCCCAATTTTGAAAACCCC (SEQ ID NO: 38)
SLC22A1	GAAGCGGCTTTGGTTGTGG (SEQ ID NO: 39)	CGCATTGCCTTTAAGACTGGC (SEQ ID NO: 40)
SLCO1B1	TTGGAGGTGTTTTGACTGCTT (SEQ ID NO: 41)	ACAAGTGGATAAGGTCGATGTTG (SEQ ID NO: 42)

Example 1. Establishment of Fah ^-/- Rag2 ^-/- IL2rg ^-/- rat using CRISPR/Cas9

To generate deletion mutations, the inventors designed sgRNAs in the second exon of rat Fah, the third exon of Rag2, and the second exon of IL2rg after analytical comparison and trial and error. The specific design is as follows (Figure 1A):

Design two sgRNAs for the second exon of Fah:

sgRNA-1: CCACGGATTGGTGTGGCCATCGG (SEQ ID NO: 1);

sgRNA-2: ATCGAAGACATGCTGATGTTTGG (SEQ ID NO: 2);

Design two sgRNAs for the third exon of Rag2:

sgRNA-1: CCTAAGAGATCCTGCCCTACTGG (SEQ ID NO: 3);

sgRNA-2: ACGAAGAGGTGGGAGGTAGCAGG (SEQ ID NO: 4);

Design two sgRNAs for the second exon of IL2rg:

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). 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.

Example 2. Fah ^-/- rats can be used as an animal model for hepatocyte transplantation and efficient reconstruction of the liver.

In the process of breeding FRG rats, 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 above results indicate that Fah ^-/- rats are a good model of hepatocyte transplantation.

Example 3, Rag2 ^{- / -} IL2rg ^{- / -} rat immune system development is seriously impaired

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. HE staining, Rag2 ^{- / -} rats deletion white pulp, IL2rg ^{- / -} rats white pulp but still significantly ^{reduced, Rag2 - / - IL2rg - /} - rats white pulp is completely absent (FIG. 3C). Simultaneous detection of immunoglobulin in serum revealed that IL2rg ^-/- rats lack IgG and contain a small amount of IgA, but the IgM content is relatively high. Rag2 ^-/- and Rag2 ^-/- IL2rg ^-/- rats were completely deficient in immunoglobulins (Fig. 4).

To further identify the immunodeficiency of Rag2 ^-/- IL2rg ^-/- rats, the inventors analyzed the population of immune cells in the thymus and spleen by flow cytometry. First, the development of T cells was examined in the thymus. CD4 ⁺ and CD8 ⁺ single positive T cells completely disappeared in Rag2 ^-/- , IL2rg ^-/- and Rag2 ^-/- IL2rg ^-/- rats relative to wild-type rats (Fig. 5A and B). CD4 ⁺ CD8 ⁺ double positive T cells almost disappeared in Rag2 ^-/- and Rag2 ^-/- IL2rg ^-/- rats, but decreased sharply in IL2rg ^-/- rats but remained (Fig. 5C).

Immediately thereafter, the inventors analyzed the development of T, B and NK cells in the spleen. 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).

The above experimental results show that Rag2 ^{- / -} IL2rg ^{- / -} rats have great defects in lymphoid organ development, immunoglobulin secretion and T, B, NK cell formation, indicating that the immune system has serious defects.

Example 4, Rag2 ^-/- IL2rg ^-/- rat can be used as a xenograft model

Immunodeficient mice are widely used for xenografts, particularly human tumor cells, embryonic stem cells, hematopoietic stem cells, and hepatocytes. To detect the degree of immunodeficiency of Rag2 ^-/- IL2rg ^-/- at the functional level, the inventors subcutaneously transplanted human hepatoma cell Snu-398 into Rag2 ^-/- IL2rg ^-/- rats and wild type rats. After 3 weeks of transplantation, all animals (n=6) developed subcutaneous tumors, but the control group (n=6) did not have any subcutaneous neoplasia (Fig. 6A and B). Subcutaneous tumor tissue was confirmed by histological analysis (Fig. 6C). Immunodeficient mice are also commonly used in teratoma formation experiments to detect pluripotency of iPS cells. The present inventors transplanted human iPS cells into Rag2 ^-/- IL2rg ^-/- rat testicular capsules, and all animals (n=3) formed teratomas after 2 months. Histological examination revealed that iPSC cells differentiated into three germ layers in vivo, including tubular epithelial cells (endoderm), plexus (ectoderm), and cartilage structure (mesoderm) (Fig. 6D).

These findings indicate that the immune system of Rag2 ^-/- IL2rg ^-/- rats is severely impaired and that these animals can xenograft human cells.

Example 5, Fah ^-/- Rag2 ^-/- IL2rg ^-/- Rats for Transplantation of Human and Mouse Primary 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. After 1.5 months of transplantation, a large amount of human albumin secretion (112-560 ug/ml) was found in the serum. At the 3 months of transplantation, the amount of albumin secretion was found to increase to 1.6 mg/ml. This shows that human hepatocytes continue to proliferate in FRG. 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%. Serial sections were subjected to human ALB protein antibody and Ki67 staining, and 91% of human hepatocytes were found to be ki67 positive (Fig. 7C). This indicates that human liver cells integrated into the rat liver are almost in a proliferative state, suggesting that human liver cells are expected to completely reconstitute the FRG liver. At the same time, by serial sectioned FAH and CYP3A4 immunohistochemical staining, human hepatocytes integrated into the liver of FRG rats expressed CYP3A4, indicating that liver humanized animals can be used to study drug metabolism (Fig. 7D).

The above results indicate that Fah ^-/- Rag2 ^-/- IL2rg ^-/- rats can be used to amplify human hepatocytes in vivo and construct liver-humanized rats.

Example 6. High proportion of liver humanized FRG rats have human liver specific metabolic gene expression and structure

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. By 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).

Further, human-specific qPCR primers showed that a high proportion of 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.

In summary, a high proportion of 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.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (18)

1. A method for producing an immunodeficient rat, comprising: disrupting a recombinant activating gene 2 and an interleukin 2 receptor γ gene; thereby obtaining an immunodeficient rat.
2. A method for preparing a rat cell, comprising: disrupting a rat recombinant activating gene 2 and an interleukin 2 receptor γ gene; preferably, the cell is a fertilized egg.
3. The method of claim 1 or 2, further comprising: disrupting the rat fumarate acetoacetate hydrolase gene.
4. The method according to claim 3, wherein the second exon of the fumarate acetylacetate hydrolase gene is disrupted; the third exon of the recombinant activator gene 2 is disrupted; and the second gene that disrupts the interleukin 2 receptor gamma gene Exon; preferably, the disruption is performed using the Crispr/Cas9 gene editing method.
5. The method according to claim 4, wherein the second exon of the fumarate acetylacetate hydrolase gene is disrupted by using sgRNA targeting the second exon of the fumarate acetylacetate hydrolase gene and Cas9 mRNA Preferably, the sgRNA is an sgRNA of the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
6. The method according to claim 4, wherein the third exon of the recombinant activating gene 2 is disrupted by using an sgRNA targeting the third exon of the recombinant activating gene 2 and Cas9 mRNA; preferably, The sgRNAs are sgRNAs of the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.
7. The method according to claim 4, wherein the second exon of the interleukin 2 receptor γ gene is disrupted by using sgRNA targeting the second exon of the interleukin 2 receptor γ gene and Cas9 mRNA; Preferably, the sgRNA is an sgRNA of the nucleotide sequence set forth in SEQ ID NO: 5 and SEQ ID NO: 6.
8. The method according to any one of claims 5 to 7, wherein said sgRNA and Cas9 mRNA or a construct capable of forming said sgRNA and Cas9 mRNA are introduced into a fertilized egg of a rat; Obtained immunodeficient rats.
9. sgRNA for the preparation of immunodeficient rats, characterized in that it targets the third exon of recombinant activator gene 2, which is the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: sgRNA; and

   It targets the second exon of 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.
10. The sgRNA according to claim 9, further comprising: an sgRNA targeting the second exon of the fumarate acetylacetate hydrolase gene, which is represented by SEQ ID NO: 1 and SEQ ID NO: The sgRNA of the nucleotide sequence.
11. A kit for preparing an immunodeficient rat, characterized in that it comprises:

    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.
12. The kit according to claim 11, further comprising: sgRNA of the nucleotide sequence shown by SEQ ID NO: 1 and SEQ ID NO: 2.
13. The kit according to claim 11, which further comprises Cas9 mRNA or a construct capable of forming Cas9 mRNA.
14. A method for preparing a rat transplantation model, comprising:

    (1) preparing an immunodeficient rat by the method according to any one of claims 1 or 1, 3 to 8;

    (2) In the immunodeficient rats of (1), heterologous cells were transplanted, and a rat transplantation model was obtained.
15. The method according to claim 14, wherein said heterologous cells comprise: hepatocytes, tumor cells, stem cells, iPS cells, blood cells.
16. The method of claim 14 wherein said heterologous cells are of human origin or of mammalian origin.
17. Use of a rat transplantation model obtained by the method of any one of claims 14 to 16 for:

    Perform drug metabolism and toxicology testing;

    Conduct liver disease and tumor research and drug screening; or

    Human hepatocytes are produced as bioreactors.
18. The use according to claim 17, wherein the liver disease comprises a hepatitis virus infection disease or malaria.

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