A fah gene knockout mouse in which fah gene expression can be restored in specific cells, and a method for preparing the same and use thereof

Abstract

The present application relates to the field of life science, in particular to a Fah gene knockout tool mouse which can restore the expression of Fah gene in specific cells and a preparation method and use thereof, the Fah gene knockout tool mouse can restore the expression of Fah gene in specific cells after Cre-loxp homologous recombination, the present application can restore the expression ability of Fah gene in specific cells such as bile duct cells of a Fah gene systemic knockout tool mouse, can induce the potential of transdifferentiation of the specific cells to hepatocytes, and can be used for screening the effect of related drugs on the transdifferentiation.

Description

Technical Field

[0001] This invention relates to the field of life sciences, and in particular to a Fah gene knockout mouse that can restore Fah gene expression in specific cells, its preparation method, and its uses. Background Technology

[0002] Fumarate acetoacetate hydrolase (Fah) is an enzyme required for the final step of the tyrosine metabolism pathway. A deficiency in the Fah gene causes hereditary tyrosinemia type I (HT1), resulting in severe damage to liver and kidney tissues. Fah knockout mice (Fah- / -) exhibit severe liver damage, with inhibited hepatocyte proliferation and increased apoptosis. Furthermore, continuous administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) effectively improves the liver and kidney damage symptoms in Fah- / - mice; discontinuation of NTBC treatment re-induces liver and kidney damage. Fah- / - mice have been widely used in liver disease research. Liver damage repair primarily relies on hepatocyte self-renewal. In recent years, scientists have discovered that under inhibited hepatocyte proliferation, bile duct epithelial cells can differentiate into hepatocytes to participate in liver damage repair. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a Fah gene knockout mouse that can restore Fah gene expression in specific cells, as well as its preparation method and uses, in order to solve the problems in the prior art.

[0004] To achieve the above and other related objectives, the present invention provides a transgenic tool mouse, which is a Fah gene knockout tool mouse. After Cre-loxp homologous recombination, the Fah gene knockout tool mouse restores the ability to express the Fah gene in specific cells.

[0005] The present invention also provides a model animal, which is the offspring obtained by mating a transgenic tool mouse with a Cre tool mouse.

[0006] The present invention also provides a method for constructing the model animal, wherein the method comprises mating the transgenic tool mouse with the Cre tool mouse to obtain the animal model.

[0007] This invention also provides a method for constructing the aforementioned transgenic tool mouse, the method comprising the following steps:

[0008] 1) The loxp-stop-loxp expression cassette was transfected into embryonic stem cells, and resistant clones were obtained after screening. Positive clones were then identified.

[0009] 2) After amplifying the positive clone, it was injected into a mouse blastocyst to obtain a chimeric mouse. The chimeric mouse was then mated with a wild-type mouse to obtain a Fah gene knockout tool mouse.

[0010] The present invention also provides the use of the transgenic tool mouse or the model animal in the preparation of animal models for drug screening.

[0011] As described above, the Fah gene knockout mouse of the present invention, which can restore Fah gene expression in specific cells, its preparation method, and its uses, have the following beneficial effects: The present invention can restore Fah gene expression in specific cells of Fah gene knockout mice, such as bile duct cells, and can induce the potential of this type of cell to transdifferentiate into hepatocytes, and can be used to screen related drugs for their effects on this transdifferentiation. The present invention greatly expands the application scope of genetic targeted manipulation and provides a valuable tool for studying cell potential transformation and drug screening in liver regeneration. Attached Figure Description

[0012] Figure 1The diagram shows the construction and validation of Fah-LSL mice, including: (a) a diagram of the Fah-LSL mouse construction strategy, in which a loxp-stop-loxp sequence is inserted into the first intron of the Fah gene. (b) a schematic diagram showing that after mating Fah-LSL and ACTB-Cre mice, Cre-loxp homologous recombination occurs, excising the stop element in Fah-LSL. (c) qRT-PCR experiments were performed to detect the expression of the Fah gene in the livers of Fah-LSL / + (i.e., mice that have recovered Fah gene expression in cells), Fah-LSL / LSL, and ACTB-Cre mice; the results showed that there was no Fah gene expression in the livers of Fah-LSL / LSL mice, while the Fah gene was normally expressed in the livers of Fah-LSL / + and ACTB-Cre mice. (d) Western spectroscopy was used to detect the expression of FAH protein in the livers of Fah-LSL / +, Fah-LSL / LSL, and ACTB-Cre mice; the results showed that no FAH protein was expressed in the livers of Fah-LSL / LSL mice, while FAH protein was normally expressed in the livers of Fah-LSL / + and ACTB-Cre mice. (e) Immunohistochemical staining was used to detect the expression of FAH protein in the livers of Fah-LSL / +, Fah-LSL / LSL, and ACTB-Cre mice; the results showed that no FAH protein was expressed in the livers of Fah-LSL / LSL mice, while FAH protein was normally expressed in the livers of Fah-LSL / + and ACTB-Cre mice. Scale bar: 100 micrometers.

[0013] Figure 2The following diagram illustrates the phenotypic verification of the Fah-LSL mice of this invention: (a) Schematic diagram of the experimental strategy: 8-week-old Fah-LSL / LSL mice were subjected to NTBC withdrawal treatment, and liver tissue was collected for analysis 6 weeks after withdrawal. (b) Weekly body weight measurements of Fah-LSL / + and Fah-LSL / LSL mice after NTBC withdrawal; mouse body weight was normalized to 1 at NTBC withdrawal. (c) H&E staining results of livers of Fah-LSL / +, Fah-LSL / LSL, and ACTB-Cre 6 weeks after NTBC withdrawal; Fah-LSL / LSL mice and Fah-LSL / LSL mice without NTBC withdrawal; the results showed that only the liver tissue of Fah-LSL / LSL mice 6 weeks after NTBC withdrawal showed cell necrosis and inflammatory cell infiltration. (d) Immunofluorescence results of liver GS (molecular marker of hepatocytes in the central venous region) and E-cad (molecular marker of hepatocytes in the portal venous region) in Fah-LSL / +, Fah-LSL / LSL, and ACTB-Cre mice 6 weeks after NTBC withdrawal; the results showed that metabolic regional disorder was only observed in the liver tissue of Fah-LSL / LSL mice 6 weeks after NTBC withdrawal. (e) Immunofluorescence staining results of liver p21 antibody in Fah-LSL / +, Fah-LSL / LSL, and ACTB-Cre mice 6 weeks after NTBC withdrawal; the results showed that p21 protein expression was only upregulated in the liver tissue of Fah-LSL / LSL mice 6 weeks after NTBC withdrawal. Scale bar: 100 micrometers.

[0014] Figure 3This invention utilizes Fah-LSL mice to induce the transdifferentiation of bile duct epithelial cells into hepatocytes, as shown in the following diagram: (a) Schematic diagram of the working principle of CK19-CreER;R26-tdT;Fah-LSL / LSL mice. After tamoxifen treatment, Cre homologous recombinase can cleave the stop element in Fah-LSL and R26-tdT, restoring the expression of the Fah gene and the expression of tdT fluorescent protein. (b) Schematic diagram of the experimental strategy. CK19-CreER;R26-tdT;Fah-LSL / LSL mice were injected with tamoxifen or corn oil (control group). Two weeks later, NTBC treatment was stopped. After 28 days of NTBC treatment, the mice were re-administered NTBC for 5 days. Liver samples were collected for analysis after 37 days. (c) Bright-field and fluorescence imaging of the whole liver sample from tamoxifen-treated CK19-CreER;R26-tdT;Fah-LSL / LSL mice 70 days after NTBC treatment was stopped. (d) Immunofluorescence staining results of tdT, CK19, and HNF4a in liver samples from CK19-CreER; R26-tdT; Fah-LSL / LSL mice 10 weeks after NTBC withdrawal in the tamoxifen-treated and oil-treated groups; the results showed that tdT-positive hepatocytes appeared in the tamoxifen-treated group, while no tdT-positive hepatocytes appeared in the oil-treated group; statistical results showed that tdT in the tamoxifen-treated group... + Bile duct cells account for about 50%, tdT + The hepatocyte count was around 25%, while the oil-treated group had no tdT. + Cells. (e) Immunofluorescence staining results of tdT and FAH in the two groups of liver samples above; the results showed that tdT in the tamoxifen group... + Cells could express FAH protein normally, while no FAH protein expression was detected in the oil-treated group. (f) ALT and AST results of NTBC in mice in the tamoxifen and oil-treated groups 10 weeks after drug withdrawal; the results showed that liver damage was reduced in the tamoxifen-treated group. Scale bar is 100 micrometers. Detailed Implementation

[0015] This invention constructs a Fah gene knockout tool mouse capable of restoring Fah gene expression in specific cells. It achieves the effect of having Fah gene expression in specific cell types while knocking out the Fah gene in other mouse tissues or cells. For example, the ability to express the Fah gene in bile duct epithelial cells can further induce the potential of bile duct epithelial cells to transdifferentiate into hepatocytes, greatly expanding the application scope of genetic targeted manipulation and providing a valuable tool for studying cellular potential transformation and drug screening in liver regeneration.

[0016] This invention provides a transgenic tool mouse, which is a Fah gene knockout tool mouse. After Cre-loxp homologous recombination, the Fah gene knockout tool mouse can restore the ability to express the Fah gene in specific cells.

[0017] The Fah gene knockout tool mouse has a loxp-stop-loxp expression cassette inserted into its Fah gene; this tool mouse can also be called a Fah-LSL / LSL mouse (Fah-LSL mouse). The Fah gene knockout tool mouse performs systemic Fah gene knockout at the genomic level and cannot express the Fah gene and Fah protein normally.

[0018] The Fah gene knockout tool mouse has a loxp-stop-loxp expression cassette inserted into the first intron of the Fah gene. In one embodiment, the loxp sequence in the loxp-stop-loxp expression cassette is a 34 bp sequence, consisting of 13 bp inverted repeats on the left and right, and an 8 bp spacer sequence in the middle. The 13 bp on the left and right are the recognition sites for Cre recombinase, and the 8 bp in the middle determines the directionality of the loxp. In one embodiment, the loxp sequence is:

[0019] ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT (SEQ ID NO: 1).

[0020] The loxp-stop-loxp expression box includes the loxp-stop-loxp sequence.

[0021]

[0022] In one embodiment, the loxp-stop-loxp expression frame further includes a homologous arm and a selection gene.

[0023] The homologous arm includes a 5' homologous arm and a 3' homologous arm.

[0024] The selection genes include positive selection genes and negative selection genes. The positive selection gene is the neo gene. The negative selection gene is MC1-TK.

[0025]

[0026] The method for inserting the loxp-stop-loxp expression cassette into the Fah gene knockout tool mouse is ES cell targeting. ES cell targeting is a conventional technique in the art and is not specifically limited herein. In one embodiment, the ES cell targeting method is as follows: constructing a targeting vector containing a homologous arm, loxp-stop-loxp, and a selection marker; linearizing the vector and transfecting it into embryonic stem cells; obtaining resistant clones after selection; and then identifying positive clones with correct homologous recombination; expanding the positive embryonic stem cell clones and injecting them into mouse blastocysts to obtain chimeric mice; mating the chimeric mice with wild-type mice and identifying them to obtain Fah gene knockout tool mice.

[0027] The Fah gene knockout tool mice undergo Cre-loxp homologous recombination through mating with Cre tool mice. Combining Fah-LSL mice with Cre tool mice, such as tissue-specific Cre or CreER mice, can induce Cre-loxp homologous recombination in specific cell types, excising the stop sequence and restoring the expression of the Fah gene and FAH protein.

[0028] In one embodiment, after the Fah gene knockout tool mouse and the Cre tool mouse are mated, Cre-loxp homologous recombination is induced by an inducer.

[0029] The inducer is an estrogen. In one embodiment, the inducer is, for example, tamoxifen.

[0030] The Cre tool mouse refers to a mouse species that can express at least Cre recombinase or a variant thereof.

[0031] Cre tools mice can be purchased commercially or built by yourself.

[0032] The Cre tool mouse is selected from systemic Cre tool mice, tissue-specific Cre tool mice, and / or inducible Cre tool mice. In a preferred embodiment, the Cre tool mouse is an inducible tissue-specific Cre tool mouse.

[0033] Tissue-specific Cre tool mice: expressed by tissue-specific promoters; for example, Alb-Cre.

[0034] Inducible Cre tool mice: By fusing Cre recombinase with the ligand-binding domain (LBD) of the hormone receptor, a fusion protein located in the cytoplasm is formed. Only after hormone induction will the fused Cre protein dissociate from the anchoring protein HSP90 through a conformational change, enter the nucleus, recognize the loxP site, and undergo recombination. For example, estrogen-induced mice.

[0035] In a preferred embodiment, the Cre tool mouse is selected from induced Cre tool mice. Preferably, it is selected from estrogen-induced Cre tool mice.

[0036] In one embodiment, the Cre tool mouse is selected from tissue-specific Cre tool mice. The tissue-specific Cre tool mouse is selected from liver-specific Cre tool mice.

[0037] In one embodiment, the Cre tool mouse is selected from liver tissue-specific and estrogen-inducible Cre tool mice.

[0038] In one embodiment, the Cre tool mouse is a CreER tool mouse. The CreER tool mouse expresses Cre recombinase and estrogen receptor (ER). Specifically, the CreER tool mouse expresses CreER in the bile duct epithelial cells of the liver.

[0039] The Fah-LSL / LSL tool mice were mated with CreER tool mice to obtain CreER mice. Fah-LSL / LSL mice underwent Cre-loxp homologous recombination under estrogen induction, which removed the stop sequence in the loxp-stop-loxp sequence of the Fah-LSL / LSL tool mice, thereby restoring the expression ability of the Fah gene in bile duct epithelial cells. After transdifferentiation into hepatocytes, FAH protein can be expressed. According to existing technology, the bile duct epithelial cells of this transgenic tool mouse can transdifferentiate into hepatocytes, thereby rescuing damaged liver function.

[0040] The "specific" cell can be any cell in the transgenic tool mouse, such as bile duct epithelial cells. The specific cells in which Fah gene expression is restored depend on the specificity of the chosen tissue-specific Cre tool mouse.

[0041] The transgenic tool mouse is a rat or a mouse.

[0042] The present invention also provides a model animal, which is the offspring obtained by mating the transgenic tool mouse with the Cre tool mouse. The offspring can express the Fah gene in specific cells, while other cells or tissues do not express the Fah gene.

[0043] The present invention also provides a method for constructing the model animal, wherein the method comprises mating the transgenic tool mouse with the Cre tool mouse to obtain the animal model.

[0044] This invention also provides a method for constructing the transgenic tool mouse, the method comprising the following steps:

[0045] 1) The loxp-stop-loxp expression cassette was linearized and transfected into embryonic stem cells. After screening, resistant clones were obtained, and positive clones with correct homologous recombination were obtained through identification.

[0046] 2) After amplifying the positive clone, it was injected into a mouse blastocyst to obtain a chimeric mouse. The chimeric mouse was then mated with a wild-type mouse to obtain a Fah gene knockout tool mouse.

[0047] The present invention also provides the use of the transgenic tool mouse in the preparation of model animals for drug screening.

[0048] The type of drug varies depending on the tissue or cell in which the Fah gene is expressed. For example, if restoring Fah gene expression in bile duct cells induces the transdifferentiation of bile duct cells into hepatocytes, the transgenic tool mouse can be used to screen for liver therapeutic or diagnostic drugs, or to screen for drugs that affect the transdifferentiation of bile duct epithelial cells into hepatocytes. The liver therapeutic drug can be a liver injury treatment drug or a liver regeneration drug.

[0049] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0050] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0051] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0052] This invention provides a Fah gene knockout mouse – Fah-LSL – capable of restoring Fah gene expression in specific cells. This genetic tool mouse has a loxp-stop-loxp sequence inserted into its first intron. The loxp-stop-loxp sequence used herein is well known in the art. The loxp sites can be recognized and cleaved by Cre recombinase. Cre-loxp homologous recombination mediates specific recombination between two loxp sites (sequences), resulting in deletion or recombination of the gene sequence between the loxp sites. The Cre-loxp homologous recombination reaction described herein will excise the stop sequence between two loxp sites, restoring the expression capacity of the Fah gene.

[0053] I. Materials and Methods

[0054] 1. Experimental materials:

[0055] Reagents: RFP antibody, FAH antibody, E-CAD antibody, GS antibody, p21 antibody, CK19 antibody, HNF4a antibody, DAPI, tamoxifen, paraformaldehyde (PFA), sucrose, PBS buffer, OCT embedding agent, donkey serum, Triton X-100, Trizol, Takara reverse transcription kit, SYBR Green, hematoxylin, eosin, RIPA protein lysis buffer, protein loading buffer, and SDS-PAGE precast gel were purchased commercially.

[0056] 2. Immunohistochemistry: The extracted liver tissue was fixed in 4% PFA for 1 hour, then washed three times with PBS for 5 minutes each time, and images were taken using a ZEISS stereomicroscope (AXIO Zoom.V16). The tissue was then dehydrated overnight in 30% sucrose solution and finally embedded with OCT. 10μm frozen sections were air-dried at room temperature, washed twice with PBS for 5 minutes each time, and blocked with PBS containing 5% donkey serum and 0.1% Triton X-100 for 30 minutes at room temperature. The primary antibody was diluted with PBS containing 2.5% donkey serum and 0.1% Triton X-100 and incubated overnight at 4°C. The slides were then washed with PBS and incubated with the corresponding secondary antibody for half an hour at room temperature. The secondary antibody was also diluted with PBS containing 2.5% donkey serum and 0.1% Triton X-100. The secondary antibody was then washed away with PBS, and the slides were blocked with mounting medium containing DAPI. Finally, images were taken using a laser confocal microscope.

[0057] 3. RNA extraction, reverse transcription, and real-time quantitative PCR

[0058] Liver tissue was collected and homogenized with 1 ml of Trizol. The homogenate was then centrifuged at 12000 g for 10 minutes, and the supernatant was transferred to a new centrifuge tube. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 seconds. After standing at room temperature for 3 minutes, the mixture was centrifuged at 12000 g for 15 minutes. The supernatant was then collected and transferred to a new centrifuge tube. 500 μL of isopropanol was added to each tube, and the mixture was inverted to mix. After standing at room temperature for 10 minutes, the mixture was centrifuged at 12000 g for 10 minutes, and the supernatant was discarded. 1 mL of 75% ethanol was added to each tube, and the mixture was inverted to mix. The tube was centrifuged at 7500 g for 5 minutes, and the supernatant was discarded. The tubes were then air-dried at room temperature. The extracted RNA was dissolved in RNAase-free water, and the RNA concentration was measured using a NanoDrop instrument. The RNA samples were stored at -80°C. All centrifugation was performed at 4°C.

[0059] According to the Takara reverse transcription kit (PrimerScript) TM The RT reagent kit with gDNA Eraser reverse transcribed 1 μg of RNA into cDNA.

[0060] Real-time PCR was then performed using a 10 μL system, with SYBR Green mix as the reagent and a QuanStudio 6 Real-Time PCR System as the instrument.

[0061] 4. Protein imprinting

[0062] Liver tissue was collected and lysed on ice for 30 minutes with RIPA protein lysis buffer, followed by centrifugation at 12000g for 10 minutes. The supernatant was collected. Protein concentration was determined, and the sample was removed, thawed on ice, and then 5×SDS loading buffer was added. The sample was boiled in water for 10 minutes to fully denature the proteins. The electrophoresis gel was removed, placed in electrophoresis buffer, and loaded. Electrophoresis was typically performed at 80-100V for 2-3 hours. Electrophoresis was stopped when bromophenol blue appeared, and membrane transfer was performed at 60-80V for 2 hours. The membrane was removed, moistened with TBS (Tween dissolved in PBS), transferred to a petri dish containing blocking buffer, and placed on a shaker for 1 hour at room temperature. The primary antibody was diluted with antibody diluent and added to a small tank. Appropriately sized bands were cut according to the marker and placed in the small tank. The mixture was incubated overnight at 4°C, followed by three 10-minute washes with TBST at room temperature. The secondary antibody was added to the small tank and incubated at room temperature for 1-2 hours, followed by three 10-minute washes with TBST at room temperature. Then a chemiluminescent reaction is performed, followed by development and photography.

[0063] Example 1 Mouse Construction

[0064] Using ES cell targeting, the loxp-stop-loxp expression cassette (e.g., ...) was knocked into intron 1 of the Fah gene. Figure 1 (As shown in a). The brief procedure is as follows: An ES cell targeting vector was constructed using infusion. This vector contains a 3.0kb 5' homologous arm, loxp-stop-loxp, PGK-Neo-polyA, a 3.0kb 3' homologous arm, and an MC1-TK-polyA negative selection marker. The nucleotide sequence of this vector is shown in SEQ ID NO: 3. After linearization, the vector was digested with enzymes and transfected into c57 and 129 hybrid F1 generation ES cells via electroporation. After screening with G418 and Ganc drugs, a total of 192 resistant clones were obtained. Long-fragment PCR identification was performed: using primers 1-ggcggggctttgggtcttg (SEQ ID NO: 4) and 2-gcgaactgctgcacgggtatc (SEQ ID NO: 5), the 5' homologous arm produced a 4.0 kb fragment; using primers 3-ttgggcagctggctaggtttttac (SEQ ID NO: 6) and 4-tgtgtttatccccagccaatcag (SEQ ID NO: 7), the 3' homologous arm produced a 4.1 kb fragment. A positive result for recombination with both homologous arms was considered a positive clone; a total of 5 positive clones with correct homologous recombination were obtained. After amplification, the positive ES cell clones were injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice. Four F1 generation positive Neo mice were obtained by mating high-proportion chimeric mice with C57BL / 6J mice.

[0065] Fah-LSL / LSL homozygous mice were obtained by mating male and female Fah-LSL mice, and the Fah gene in these mice was deleted. Figure 2 As shown, mice can maintain a normal physiological state under NTBC treatment, but severe liver damage occurs when NTBC treatment is stopped.

[0066] ACTB-Cre mice were obtained by mating Fah-LSL and commercially available ACTB-Cre mice; Fah-LSL / LSL mice and ACTB-Cre mice expressed Cre recombinase throughout their cells, and the stop sequence in the loxp-stop-loxp sequence of Fah-LSL / LSL mice was removed (e.g., ...). Figure 1 (As shown in b). Figure 1 As shown in C to E, ACTB-Cre;Fah-LSL / LSL mice restored the expression of the cellular Fah gene. Mice that restored the expression of the cellular Fah gene can be denoted as Fah-LSL / +.

[0067] Example 2: A model for inducing transdifferentiation of bile duct epithelial cells into hepatocytes in Fah-LSL mice.

[0068] To verify whether Fah-LSL / LSL mice can induce the transdifferentiation of bile duct epithelial cells into hepatocytes, this example used CK19-CreER, R26-tdT mice (both purchased from The Jackson Laboratory) and Fah-LSL / LSL mice to obtain CK19-CreER;R26-tdT;Fah-LSL / LSL mice. Tamoxifen induction in these mice could label bile duct cells with tdT. + Simultaneously, the stop sequence in the Fah gene is excised from bile duct epithelial cells and their progeny cells. Then, proceed as follows... Figure 3 Strategy b was used to induce liver injury in mice by discontinuing NTBC treatment. To improve mouse survival, NTBC treatment was resumed for 5 days midway through the experiment. Liver tissue from mice was collected 10 weeks after NTBC discontinuation for whole-specimen fluorescence and bright-field imaging experiments. The results are shown in Figure b. Figure 3 As shown in Figure c, tdT is present in the liver. + Clump signal. Further frozen section and tdTCK19 and HNF4a immunohistochemical experiments, such as Figure 3 As shown in Figure d, numerous tdT cells were observed in the livers of CK19-CreER;R26-tdT;Fah-LSL / LSL mice treated with tamoxifen. + Hepatocytes, specifically bile duct epithelial cells, underwent transdifferentiation into hepatocytes, and statistical results showed tdT... + Hepatocytes account for approximately 25% of the total hepatocytes; however, tdT was not observed in the livers of CK19-CreER;R26-tdT;Fah-LSL / LSL mice that were not induced by tamoxifen. + Hepatocytes, i.e., those that have not undergone transdifferentiation from bile duct epithelial cells into hepatocytes. In addition, such as... Figure 3 As shown in Figure e, the FAH immunofluorescence staining experiment showed tdT + Hepatocytes all express FAH. For example... Figure 3 As shown in Figure f, compared with the control group, the transdifferentiation of bile duct epithelial cells into hepatocytes reduced ALT and AST levels, alleviating liver damage. These results indicate that in the Fah-LSL mouse model, restoring Fah gene expression in bile duct epithelial cells can induce their transdifferentiation into hepatocytes.

[0069] This invention provides a Fah gene knockout mouse capable of restoring Fah gene expression in specific cells. The invention constructs Fah-LSL mice by inserting a loxp-stop-loxp sequence into the Fah gene, preventing normal Fah gene expression. Combining Fah-LSL mice with tissue-specific CreER mice allows Cre-loxp homologous recombination to occur in specific cell types, excising the stop sequence and restoring Fah protein expression. Using CK19-CreER;R26-tdT;Fah-LSL / LSL mice as an example, this invention specifically illustrates how to apply this system to induce the transdifferentiation of bile duct epithelial cells into hepatocytes. Subsequently, CK19-CreER;R26-tdT;Fah-LSL / LSL mice can be used to study the regulatory effects of small molecules and drugs on this transdifferentiation. Furthermore, Fah-LSL mice can be used to study the transdifferentiation of other cell types into hepatocytes. This technology greatly expands the application scope of genetic targeted manipulation and provides a valuable tool for liver regenerative medicine research.

[0070] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for constructing an animal model in which expression of a Fah gene is restored in specific cells, characterized by, The construction method includes the following steps: 1) The loxp-stop-loxp expression cassette was transfected into mouse embryonic stem cells so that the first intron of the Fah gene of the embryonic stem cells was inserted with the loxp-stop-loxp expression cassette. After screening, resistant clones were obtained, and positive clones with correct homologous recombination were obtained by identification. 2) After amplifying the positive clone, it is injected into a mouse blastocyst to obtain a chimeric mouse. The chimeric mouse is then mated with a wild-type mouse to obtain a Fah gene knockout tool mouse. The Fah gene knockout tool mouse undergoes systemic Fah gene knockout at the genomic level. 3) The animal model was obtained by mating the Fah gene knockout tool mouse and the CreER tool mouse; In this process, the Fah gene knockout tool mouse and the CreER tool mouse are mated and undergo Cre-loxp homologous recombination to restore the ability to express the Fah gene in specific cells, namely, the bile duct epithelial cells of the liver. The CreER tool mouse expresses CreER in the bile duct epithelial cells of the liver. The Fah gene knockout tool mouse obtains CreER by mating with the CreER tool mouse. In Fah-LSL / LSL mice, under the induction of estrogen, Cre-loxp homologous recombination occurs, and the stop sequence in the loxp-stop-loxp sequence is removed, thereby restoring the expression ability of the Fah gene in the bile duct epithelial cells. Then, the bile duct epithelial cells with restored Fah gene expression ability transdifferentiate into hepatocytes and express Fah protein.

2. The method for constructing an animal model that restores Fah gene expression in specific cells according to claim 1, characterized in that, The loxp-stop-loxp expression frame also includes homologous arms and selection genes.

3. The method for constructing an animal model that restores Fah gene expression in specific cells according to claim 2, characterized in that, The nucleotide sequence of the loxp-stop-loxp expression cassette is shown in SEQ ID NO:

3.

4. The use of the animal model constructed by the method for constructing an animal model that restores Fah gene expression in specific cells as described in any one of claims 1-3 in drug screening.

5. The use according to claim 4, wherein the drug is a liver treatment drug.

6. The liver treatment drug according to claim 5 is a liver injury treatment drug.

7. The liver treatment drug according to claim 5 is a liver regeneration drug.

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