Monoclonal cell strain for knocking out Tan sheep FABP4 gene as well as construction method and application of monoclonal cell strain

Abstract

The invention provides a construction method of a monoclonal cell strain for knocking out a Tan sheep FABP4 gene, which comprises the following steps: knocking out the Tan sheep FABP4 gene by using a CRISPR / Cas9 system, targeting a first exon of the Tan sheep FABP4 gene by using the CRISPR / Cas9 system, and showing a nucleotide sequence of the first exon of the Tan sheep FABP4 gene as SEQ ID NO.1; sgRNA in the CRISPR / Cas9 system targets the nucleotides at the 2-8 sites at the 5'end in the sequence SEQ ID NO.1, and the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.2; the method comprises the following steps: constructing a Lentiti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid, and carrying out lentivirus packaging, drug screening and monoclonal screening, so as to obtain the monoclonal cell strain of which the Tan sheep FABP4 gene is knocked out. The invention also provides application of the monoclonal cell strain, and the monoclonal cell strain is used for constructing a cell model of a Tan sheep precursor fat cell differentiation regulation mechanism. According to the invention, the Tan sheep FABP4 gene is knocked out by using a CRISPR / Cas9 system, so that the research on the function and regulation mechanism of the FABP4 gene in Tan sheep precursor fat cell differentiation is facilitated, and a new molecular target is provided for controlling excessive fat accumulation.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular biology technology, specifically relating to a single-clonal cell line with the FABP4 gene knocked out from Tan sheep, its construction method, and its application. Background Technology

[0002] Tan sheep are a unique long-fat-tailed breed in my country, with adult ewes having a tail fat weight of 6-8 jin (approximately 3-4 catties), accounting for about 10% of their body weight. However, with the improvement of modern living standards and feeding conditions, the market demand and commercial value of Tan sheep fat have significantly decreased. More importantly, excessive tail fat deposition not only reduces carcass yield but also increases breeding costs due to excessive subcutaneous fat accumulation, thus affecting economic benefits. Against this backdrop, how to reduce tail fat deposition while maintaining its excellent stress resistance traits through genetic improvement has become a crucial issue that urgently needs to be addressed in the current Tan sheep breeding field.

[0003] Fatty acid binding protein 4 (FABP4) is a member of the fatty acid binding protein family and plays a central role in regulating fatty acid metabolism, transport, and lipid synthesis and breakdown. Studies have shown that the expression level of the FABP4 gene is closely related to fat deposition in animals, and knocking out or inhibiting this gene can reduce fat accumulation. Currently, there is limited research on the function of the FABP4 gene in Tan sheep, especially lacking a systematic approach to constructing a FABP4-deficient Tan sheep preadipocyte model using gene editing technology to explore its role in the regulation of fat deposition.

[0004] CRISPR / Cas9 is an acquired immune defense system developed by bacteria during long-term evolution, capable of resisting the invasion of foreign viruses or genetic material. It has been widely and effectively applied in cell gene modification technology. With further development and application, gene knockout technology, as the first step in molecular basic research in animal husbandry, is widely used to clarify the evolutionary and regulatory mechanisms of gene function, providing a theoretical basis for animal breeding and production development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a single-clone cell line with the FABP4 gene knocked out in Tan sheep, its construction method and application, in order to address the shortcomings of the prior art. This method helps to study the role of the FABP4 gene in the differentiation of Tan sheep preadipocytes and provides a new molecular target for controlling excessive fat accumulation.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for constructing a monoclonal cell line with the FABP4 gene knocked out of Tan sheep, wherein the FABP4 gene of Tan sheep is knocked out using the CRISPR / Cas9 system, wherein the CRISPR / Cas9 system targets the first exon of the FABP4 gene of Tan sheep, and the nucleotide sequence of the first exon is shown in SEQ ID NO.1.

[0007] Preferably, the sgRNA in the CRISPR / Cas9 system targets nucleotides 2 to 8 at the 5' end of the sequence SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.2.

[0008] Preferably, the method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep is as follows: S1. Synthesize the sgRNA; S2. Design and synthesize specific upstream and downstream primers based on the nucleotide sequence of the sgRNA; The specific upstream primers are sgRNA-F and OaFABP4-sgRNA-F, with nucleotide sequences shown in SEQ ID NO. 3-4, respectively; the specific downstream primers are sgRNA-R and OaFABP4-sgRNA-R, with nucleotide sequences shown in SEQ ID NO. 5-6, respectively. S3. Using the Lenti-CRISPR V2-Puro vector plasmid as a template, PCR amplification was performed using the specific upstream primer sgRNA-F and the specific downstream primer OaFABP4-sgRNA-R to obtain target fragment 1. PCR amplification was then performed using the specific upstream primer OaFABP4-sgRNA-F and the specific downstream primer sgRNA-R to obtain target fragment 2. S4. The Lenti-CRISPR V2-Puro vector plasmid was digested with restriction endonucleases EcoR I and Kpn I, and the linearized vector Lenti-CRISPR V2-Puro was obtained after gel recovery. S5. Ligate target fragment 1 and target fragment 2 obtained in S3 with the linearized vector Lenti-CRISPRV2-Puro obtained in S4. After extracting the plasmid, obtain the Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid. S6. The Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid and lentiviral packaging plasmid obtained in S5 were co-transfected into HEK293T cells to obtain lentiviral solution. S7. The lentivirus solution obtained in S6 was used to infect Tan sheep preadipocytes. After drug screening with puromycin hydrochloride solution, a cell line with the FABP4 gene knocked out of Tan sheep was obtained. S8. After digesting the cell line with the knockout of the Tan sheep FABP4 gene obtained in S7 with trypsin, perform single-clone screening to obtain a single-clone cell line with the knockout of the Tan sheep FABP4 gene.

[0009] Preferably, the PCR amplification reaction system described in S3 is as follows: 1 μL of sgRNA-F / OaFABP4-sgRNA-F, 1 μL of OaFABP4-sgRNA-R / sgRNA-R, 1 μL of Lenti-CRISPR V2-Puro vector plasmid, 12.5 μL of 2×Phanta MaxMaster Mix, and ddH2O to a final volume of 25 μL; the reaction program is as follows: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min.

[0010] Preferably, the double digestion reaction system in S4 is as follows: 10 μg of Lenti-CRISPR V2-Puro vector plasmid, 10 μL of restriction endonuclease EcoR I, 10 μL of Kpn I, 20 μL of 10×M Buffer, and ddH2O to a final volume of 200 μL; the double digestion reaction procedure is as follows: double digestion at 37°C for 2 h.

[0011] Preferably, the ligation system in S5 is: target fragment 1 25 ng, target fragment 2 25 ng, linearization vector Lenti-CRISPR V2-Puro 145 ng, 2×Seamless Cloning Mix 5 μL, and ddH2O to a final volume of 10 μL; the ligation procedure is: ligation at 50°C for 45 min.

[0012] Preferably, the lentiviral packaging plasmid in S6 is PSPAX2 plasmid and PMD2.G plasmid; the co-transfection time is 48 h; the infection time in S7 is 48 h; the mass concentration of the puromycin hydrochloride solution is 2 μg / mL; and the mass fraction of the trypsin in S8 is 0.25%.

[0013] This invention also provides the application of a monoclonal cell line of Tan sheep with the FABP4 gene knocked out by the above method, and the application of the monoclonal cell line with the FABP4 gene knocked out in Tan sheep in constructing a cell model of the differentiation regulation mechanism of Tan sheep preadipocytes.

[0014] Compared with the prior art, the present invention has the following advantages: The CRISPR / Cas9 system of this invention, as a powerful gene editing technology, has been widely used in the field of veterinary medicine. Knocking out the FABP4 gene in Tan sheep using the CRISPR / Cas9 system helps to study the regulatory role and mechanism of the FABP4 gene on fatty acid metabolism, differentiation and lipid droplet deposition in Tan sheep preadipocytes, and provides a new molecular target for controlling excessive fat accumulation.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is an image showing the electrophoresis results of double enzyme digestion of the pET32a-OaFABP4 recombinant plasmid in Example 1 of the present invention.

[0017] Figure 2 These are Western blot analysis diagrams of negative serum and rOaFABP4 serum as primary antibodies recognizing FABP4 protein in Example 1 of the present invention; wherein, Figure A is the Western blot analysis of normal rat serum as primary antibody recognizing FABP4 protein; Figure B is the Western blot analysis of rOaFABP4 immunized rat serum as primary antibody recognizing FABP4 protein.

[0018] Figure 3 This is a sequencing identification diagram of the Lenti-CRISPR V2-Puro-OaFABP4-sgRNA vector plasmid in Example 1 of the present invention; wherein, the red box represents the sgRNA nucleotide sequence.

[0019] Figure 4 This is a DNA detection diagram of a single-clonal cell line with FABP4 knockout genotype, as shown in Example 1 of the present invention.

[0020] Figure 5 This is a Western Blot diagram illustrating the effect of FABP4 gene knockout in a single-clonal cell line as verified in Example 1 of this invention.

[0021] Figure 6 This is a diagram illustrating the effect of FABP4 gene knockout in a monoclonal cell line verified by cell immunofluorescence in Example 1 of the present invention.

[0022] Figure 7 This is a cell viability graph of the FABP4-WT group and the FABP4-AID group at different time periods detected by the CCK-8 kit in Example 1 of the present invention.

[0023] Figure 8 This is an Oil Red O staining image of cells in the FABP4-WT group and FABP4-AID group after induced differentiation in Example 2 of the present invention.

[0024] Figure 9 This is a diagram showing the expression of PPARγ and CEBPα proteins in cells of the FABP4-WT group and the FABP4-AID group by Western Blot in Example 2 of the present invention.

[0025] Figure 10 This is a diagram showing the expression of FASN and ACC1 proteins in cells of the FABP4-WT group and the FABP4-AID group by Western Blot in Example 2 of the present invention.

[0026] Figure 11 This is a diagram showing the expression of ATGL and HSL proteins in cells of the FABP4-WT group and the FABP4-AID group by Western Blot in Example 2 of the present invention.

[0027] Figure 12 This is a graph showing the triglyceride and free fatty acid content in cells of the FABP4-WT group and the FABP4-AID group in Example 2 of the present invention. Detailed Implementation

[0028] Example 1 The method for isolating and culturing Tan sheep preadipocytes in this embodiment is as follows: The newborn Tan sheep were euthanized, and the whole sheep were disinfected with benzalkonium chloride and alcohol. Under aseptic conditions, subcutaneous adipose tissue was collected from the tail and washed in phosphate-buffered saline (PBS) containing 5% penicillin-streptomycin solution. After cleaning, the tissue was cut into small pieces with sterile scissors, and double the volume of type I collagenase was added and digested in a shaker at 37°C for 2 hours. Digestion is terminated, and the tissue is filtered through a sieve with a pore size of 45μm to remove large pieces of tissue; Cells were collected by centrifugation at 1000g for 5 minutes; Add red blood cell lysis buffer to remove red blood cells, then centrifuge and discard the supernatant; Resuspend cells in complete culture medium and at 25 cm⁻¹ 2 Cell culture flasks at 37°C, 5% CO2 2 They are cultured in a cell culture incubator.

[0029] The method for preparing rOaFABP4 protein serum in this embodiment is as follows: Specific primers were designed based on the coding region of the FABP4 gene in Tan sheep. The amino acid sequence of the FABP4 gene coding region is shown in SEQ ID NO.7. Total RNA was extracted from Tan sheep preadipocytes using Trizol reagent, and the total RNA was used to synthesize first-strand cDNA according to the instructions of the Novizan reverse transcription kit. Using the cDNA as a template, the FABP4 gene was amplified by PCR. The nucleotide sequence of the specific upstream primer OaFABP4-F for PCR amplification is shown in SEQ ID NO.8, and the nucleotide sequence of the specific downstream primer OaFABP4-R is shown in SEQ ID NO.9. The PCR amplification product was cloned and ligated into the pMD19T vector to obtain the pMD19T-OaFABP4 recombinant vector plasmid, which was then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The PCR amplification reaction system consisted of: 1 μL of specific upstream primer OaFABP4-F, 1 μL of specific downstream primer OaFABP4-R, 1 μL of cDNA template, 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), and ddH2O to a final volume of 25 μL. The reaction program was: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 24 s, for 35 cycles; 72℃ for 5 min. The ligation system consisted of: 200 ng of PCR product, 1 μL of pMD19-T vector, 5 μL of Solution I, and ddH2O to a final volume of 10 μL. The ligation program was: ligation at 16℃ for 2 h.

[0030] The FABP4 gene fragment was amplified by PCR using specific primers OaFABP4-32a-F and OaFABP4-32a-R with restriction enzyme sites as templates, with the pMD19T-OaFABP4 recombinant vector plasmid as template. The nucleotide sequence of the specific primers OaFABP4-32a-F is shown in SEQ ID NO.10, and the nucleotide sequence of OaFABP4-32a-R is shown in SEQ ID NO.11. The pET32a vector was digested with Xho I and BamHI, and the digested vector and gene fragment were ligated using T4 ligase to obtain the pET32a-OaFABP4 recombinant plasmid. Restriction digestion was performed to verify the plasmid ligation. The digestion results are shown in the figure below. Figure 1As shown, the pET32a-OaFABP4 recombinant plasmid was successfully obtained. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, positive colonies were selected, and after expansion culture, OaFABP4 protein was purified by nickel column affinity chromatography. Using 200g-220g SPF-grade SD rats as experimental subjects, polyclonal antibodies against OaFABP4 protein were prepared. Serum from rats was collected by orbital blood collection, centrifuged at 2500 rpm for 15 min under sterile conditions, and red blood cells and other cell debris were removed. After repeated centrifugation, the supernatant was collected to obtain rOaFABP4 protein serum. The PCR amplification reaction system consisted of: 1 μL of OaFABP4-32a-F, 1 μL of OaFABP4-32a-R, 1 μL of pMD19T-OaFABP4, 12.5 μL of 2×Phanta Max Master Mix (Dye Plus), and ddH2O to a final volume of 25 μL. The PCR amplification reaction program was as follows: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 24 s, for 35 cycles; 72℃ for 5 min. The reaction system for the double enzyme digestion was as follows: pET32a 10 μg, Xho I 10 μL, BamHI 10 μL, 10×KBuffer 20 μL, and ddH2O to a final volume of 200 μL; the reaction procedure for the double enzyme digestion was: double digestion at 37℃ for 2 h. The ligation reaction system consisted of 90 ng of linearized pET32a vector, 49 ng of PCR product, 10 μL of Ligation Mix, and ddH2O to a final volume of 20 μL. The ligation reaction procedure was to ligate at 16 °C for 2 h.

[0031] Sheep proadipocytes were electrophoresed using 12.5% ​​sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the cells were transferred onto polyvinylidene fluoride (PVDF) membranes using a wet transfer method. The PVDF membranes were incubated with primary antibodies (1:100) of negative serum (normal rat serum) and rOaFABP4 protein serum, and secondary antibody (1:10000) of goat anti-rat IgG. Finally, the membranes were exposed to a high-sensitivity ECL chemiluminescent substrate (ECL luminescent solution) for color development. The results are as follows: Figure 2 As shown in Figures A and B, the negative serum incubated with the primary antibody showed no specific band, while the rOaFABP4 serum incubated with the primary antibody showed a specific band at around 15 kDa, indicating that the serum has good antigenicity and can specifically recognize the FABP4 protein.

[0032] This embodiment describes a method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep. The method is as follows: S1. Using the coding region of the FABP4 gene as the target sequence, sgRNA was designed and synthesized using the CRISPR / Cas9 gene targeting tool via the E-CRISPR website. Ovis aries were selected as the species, and the sgRNA with the highest score was chosen. The CRISPR / Cas9 system targeted the first exon of the FABP4 gene of Tan sheep, and the nucleotide sequence of the first exon is shown in SEQ ID NO.1. The sgRNA targeting sequence is the nucleotides 2 to 8 at the 5' end of SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.2. S2. Design and synthesize specific upstream and downstream primers based on the nucleotide sequence of the sgRNA; The specific upstream primers are sgRNA-F and OaFABP4-sgRNA-F, with nucleotide sequences shown in SEQ ID NO. 3-4, respectively; the specific downstream primers are sgRNA-R and OaFABP4-sgRNA-R, with nucleotide sequences shown in SEQ ID NO. 5-6, respectively. S3. Using the Lenti-CRISPR V2-Puro vector plasmid as a template, PCR amplification was performed using the specific upstream primer sgRNA-F and the specific downstream primer OaFABP4-sgRNA-R to obtain target fragment 1. PCR amplification was then performed using the specific upstream primer OaFABP4-sgRNA-F and the specific downstream primer sgRNA-R to obtain target fragment 2. The PCR amplification reaction system was as follows: 1 μL of sgRNA-F / OaFABP4-sgRNA-F, 1 μL of OaFABP4-sgRNA-R / sgRNA-R, 1 μL of Lenti-CRISPR V2-Puro vector plasmid, 12.5 μL of 2×Phanta Max Master Mix (DyePlus), and ddH2O to a final volume of 25 μL. The reaction program was: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min. S4. The Lenti-CRISPR V2-Puro vector plasmid was digested with restriction endonucleases EcoRI and KpnI. After gel recovery using a gel recovery kit (White Shark Biotechnology Co., Ltd.), the linearized vector Lenti-CRISPR V2-Puro was obtained. The double digestion reaction system was as follows: 10 μg Lenti-CRISPR V2-Puro vector plasmid, 10 μL restriction endonuclease EcoRI, 10 μL KpnI, 20 μL 10×M Buffer, and ddH2O to a final volume of 200 μL; the double digestion reaction program was as follows: double digestion at 37℃ for 2 h. S5. Target fragments 1 and 2 obtained in S3 were ligated to the linearized vector Lenti-CRISPRV2-Puro obtained in S4 using white shark homologous recombinase. After transformation into E. coli DH5α competent cells, the cells were plated, and positive colonies were picked and identified by bioengineering sequencing. The results are as follows: Figure 3 As shown, the red box represents the sgRNA nucleotide sequence. After extracting the plasmid from the suitable bacterial culture using a plasmid miniprep kit (White Shark Biotechnology Co., Ltd.), the Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid was obtained. The ligation system consisted of: target fragment 1 25 ng, target fragment 2 25 ng, linearization vector Lenti-CRISPR V2-Puro 145 ng, 2×Seamless Cloning Mix 5 μL, and ddH2O to a final volume of 10 μL. The ligation procedure was as follows: ligation was performed at 50 °C for 45 min. S6. The Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid obtained in S5 was co-transfected with the lentiviral packaging plasmids PSPAX2 and PMD2.G into human embryonic kidney cells (HEK293T) with a cell density of 85% using Lipo2000. Cell supernatants were collected 48h and 72h after transfection, and the lentiviral solution was concentrated using the 5× polyethylene glycol 8000 (PEG8000) method to obtain lentiviral solution. S7, 5×10 5 One well of Tan sheep preadipocytes was seeded into a 6-well plate, and complete culture medium was added to the plate. When the cell density reached 55%, lentivirus solution obtained from S6 was added to infect the Tan sheep preadipocytes as the lentivirus infection group, and cells without lentivirus solution were used as the control group. After 48 hours of infection, puromycin hydrochloride solution with a mass concentration of 2 μg / mL was added for drug screening. When almost all cells in the control group died, puromycin hydrochloride solution was added to the lentivirus infection group until the cells reached confluence. The cells were then expanded to obtain a cell line with the FABP4 gene knocked out from Tan sheep. S8. The cell lines obtained in S7 with the FABP4 gene knocked out from Tan sheep were digested with 0.25% trypsin. The digested cell suspension was then seeded into 96-well plates using the infinite dilution method. After single-cell colonies formed, the cells were expanded to obtain monoclonal cell lines with the FABP4 gene knocked out. DNA was extracted from these monoclonal cell lines to detect the FABP4 genotype. The results are as follows: Figure 4 As shown, sequencing results indicate the presence of frameshift mutations, where "-" represents base deletion.

[0033] This embodiment also identified the FABP4 gene knockout effect of the monoclonal cell line with the FABP4 gene knocked out of Tan sheep obtained above.

[0034] The monoclonal cell line with the knockout of the FABP4 gene in Tan sheep obtained in step S8 was used as the experimental group cells, denoted as FABP4-AID; and the Tan sheep preadipocytes isolated and cultured were used as the control group cells, denoted as FABP4-WT.

[0035] (1) Verification of FABP4 gene knockout effect using Western blotting: 50% of the single-clone cell line with FABP4 gene knockout obtained in step S8 was collected, added to RIPA lysis buffer, and repeatedly frozen and thawed to disrupt the cells. After adding 5× loading buffer, the cells were heated in a 95℃ metal bath for 20 min. Subsequently, cells in the FABP4-AID group and FABP4-WT group were electrophoresed with 12.5% ​​SDS-PAGE at 80V for 20 min and 120V for 50 min. After the electrophoresis, the cells were transferred to a PVDF membrane by wet transfer at a constant current of 300mA for 30 min. The PVDF membrane was blocked with 5% skim milk for 1.5 h. Then, the cells were incubated overnight at 4℃ with rOaFABP4 protein serum as the primary antibody (1:100 dilution). The PVDF membrane was washed three times with TBST buffer on a shaker for 5 min each time. The cells were then incubated with HRP-antibody. RAT (1:10000 dilution) was used as the secondary antibody to incubate the PVDF membrane; it was washed three times with TBST buffer for 10 min each time; finally, it was placed on a Bio-Rad exposure apparatus, and ECL was added for exposure and development; the results are shown in the attached figure. Figure 5 As shown, this indicates that the FABP4 gene has been knocked out.

[0036] (2) Verification of FABP4 gene knockout effect using cell immunofluorescence: FABP4-AID and FABP4-WT cells were re-platened. When the cell density reached 65%, the cell culture medium was discarded, and the cells were washed three times with PBS. Cells were then fixed with 4% paraformaldehyde solution at room temperature for 30 min. 0.5% Triton X-100 immunostaining permeabilization buffer was added, and the cells were permeabilized at room temperature for 30 min. Then, 5% bovine serum albumin (BSA) was added, and the cells were blocked at room temperature for 1 h. The blocking solution was discarded, and the cells were incubated overnight at 4°C with primary antibody Anti-OaFABP4 protein serum (1:500 dilution). The cells were washed three times with PBS, and Cy3-labeled goat anti-rat IgG (1:10000 dilution) was added and incubated at 37°C in the dark for 1 h. The cells were washed three times with PBS, and nuclear staining was performed for 10 min with 10 µg / mL DAPI dilution. The expression of FABP4 protein in cells was observed under laser confocal microscopy. The results are as follows: Figure 6 As shown, cells in the FABP4-WT group glow red, while cells in the FABP4-AID group do not glow, indicating that the knockout was successfully verified.

[0037] (3) The effect of FABP4 gene knockout on cell proliferation rate was detected using the CCK-8 assay kit. One day in advance, cells in the FABP4-WT group and the FABP4-AID group were divided into two groups at 1×10⁻⁶ cells per ... 5 Cells were seeded at a ratio of [number] cells / well in 96-well plates, and cell proliferation activity was assessed at 24 h, 48 h, and 72 h. Results are as follows: Figure 7 As shown, knockout of the FABP4 gene has no significant effect on the proliferation of preadipocytes in Tan sheep.

[0038] Example 2 This example demonstrates the application of a single-clone cell line with the knockout of the FABP4 gene from Tan sheep in Example 1 for constructing a cell model of the differentiation regulation mechanism of Tan sheep preadipocytes. Knocking out the FABP4 gene can reduce lipid droplet deposition in Tan sheep preadipocytes; decrease the expression levels of genes related to Tan sheep preadipocyte differentiation and fatty acid metabolism; increase the expression level of triglyceride lipase genes in Tan sheep preadipocytes; and decrease the expression level of hormone-sensitive lipase genes and the content of triglycerides and free fatty acids.

[0039] (a) A single-clonal cell line with the FABP4 gene knocked out from Tan sheep was used as a cell model to study the formation of lipid droplets after precursor fat fractionation: Cells from the FABP4-WT group and the FABP4-AID group were seeded into 6-well plates. After two days of contact inhibition, differentiation induction medium was added to induce differentiation for 2 days. Then, the medium was replaced with maintenance differentiation medium, and the medium was changed every 2 days until differentiation was complete. The differentiation induction medium was prepared by adding the following raw materials to DMEM medium in the following final amounts: fetal bovine serum (FBS) 10wt% + penicillin and antibiotics 2wt% + 3-isobutyl-1-methylxanthine (IBMX) 0.5mmol / L + dexamethasone 1.0μmol / L + insulin 10μg / mL + rosiglitazone 1.0μmol / L; The differentiation maintenance medium was prepared by adding the following raw materials to DMEM medium in the following final amounts: FBS 5wt% + penicillin and anti-inflammatory drugs 2wt% + insulin 10μg / mL + rosiglitazone 1.0μmol / L.

[0040] Cells from the FABP4-WT and FABP4-AID groups after induced differentiation were stained with Oil Red O, and the staining was observed under a microscope. Results are as follows: Figure 8 As shown in Figures A and B, Figure A shows Oil Red O staining of cells in the FABP4-WT group, and Figure B shows Oil Red O staining of cells in the FABP4-AID group. Compared with the FABP4-WT group, the FABP4-AID group showed reduced lipid droplet formation.

[0041] (II) A single-clonal cell line with the FABP4 gene knocked out from Tan sheep was used as a cell model for studying precursor adipocyte differentiation: Cells from the FABP4-WT and FABP4-AID groups induced to differentiate to day 8 were collected, RIPA lysis buffer was added, and the cells were repeatedly frozen and thawed before sonication lysis. 5× loading buffer was added and the cells were boiled at 95℃ for 20 min. After brief centrifugation, the cells were stored at -20℃ for Western blot detection.

[0042] (1) Western blot analysis was performed to detect the expression of adipocyte differentiation-related genes peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (CEBPα) in the two groups of cells after induced differentiation. The results are as follows: Figure 9 As shown, compared with the FABP4-WT group, the expression levels of PPARγ and CEBPα in the FABP4-AID group were decreased, indicating that knocking out the FABP4 gene inhibits the differentiation of Tan sheep preadipocytes.

[0043] (2) Western blot was used to detect the expression of fatty acid metabolism-related genes fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1) in cells of the FABP4-WT and FABP4-AID groups after induced differentiation. The results are as follows: Figure 10 As shown, compared with the FABP4-WT group, the expression levels of FASN and ACC in the FABP4-AID group were decreased.

[0044] (3) Western blot analysis was performed on the expression of triglyceride (TAG) metabolism-related genes, acetylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL), in cells of the FABP4-WT and FABP4-AID groups after induced differentiation. The results are as follows: Figure 11 As shown, compared with the FABP4-WT group, the ATGL expression level of the FABP4-AID group was upregulated, while the HSL expression level was decreased.

[0045] (III) A single-clonal cell line with the FABP4 gene knocked out from Tan sheep was used as a cell model to study changes in triglyceride and free fatty acid content in preadipocytes: Cells from the FABP4-WT and FABP4-AID groups were seeded in 6-well plates and cultured for 2 days before inducing differentiation. The levels of triglycerides (TG) and free fatty acids (FFA) in the differentiated cells of both groups were measured using a triglyceride and free fatty acid assay kit. Results are as follows: Figure 12 As shown in Figures A and B, compared with the FABP4-WT group, the intracellular TG and FFA contents in the FABP4-AID group were significantly decreased (P < 0.01).

[0046] In summary, sequencing results confirm the successful construction of a monoclonal cell line with the FABP4 gene knocked out from Tan sheep. To investigate the effect of FABP4 on adipocyte function, this monoclonal cell line was used as a cell model. Western blotting was used to detect the expression of key genes: compared to the control group, after induction of differentiation, the expression levels of core adipocyte differentiation regulatory genes PPARγ and CEBPα in the FABP4 gene knockout group were significantly decreased, as were the expression levels of fatty acid metabolism-related genes FASN and ACC1, while the expression level of TAG metabolism-related gene ATGL increased, and the expression level of HSL decreased. Morphological validation showed that Oil Red O staining indicated a significant reduction in the number of lipid droplets formed in the FABP4 knockout group. Further analysis of TG and FFA levels confirmed that FABP4 gene knockout significantly reduced the accumulation of TG and FFA in cells. Therefore, it can be concluded that FABP4 gene knockout can directly lead to a decrease in intracellular lipid (TG, FFA) content and a reduction in lipid droplet formation by regulating the expression of key genes in fatty acid synthesis. This indicates that FABP4 gene is closely related to the normal differentiation and maturation capacity of adipocytes and lipid synthesis and storage function, and is an important regulatory factor for maintaining the physiological function of adipocytes.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep, characterized in that, The method is as follows: the FABP4 gene of Tan sheep is knocked out using the CRISPR / Cas9 system, which targets the first exon of the Tan sheep FABP4 gene. The nucleotide sequence of the first exon is shown in SEQ ID NO.

1.

2. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 1, characterized in that, The sgRNA in the CRISPR / Cas9 system targets nucleotides 2 to 8 at the 5' end of the sequence SEQ ID NO.1, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.

2.

3. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 1, characterized in that, The method is as follows: S1. Synthesize the sgRNA; S2. Design and synthesize specific upstream and downstream primers based on the nucleotide sequence of the sgRNA; The specific upstream primers are sgRNA-F and OaFABP4-sgRNA-F, with nucleotide sequences shown in SEQ ID NO. 3-4, respectively; the specific downstream primers are sgRNA-R and OaFABP4-sgRNA-R, with nucleotide sequences shown in SEQ ID NO. 5-6, respectively. S3. Using the Lenti-CRISPR V2-Puro vector plasmid as a template, PCR amplification was performed using the specific upstream primer sgRNA-F and the specific downstream primer OaFABP4-sgRNA-R to obtain target fragment 1. PCR amplification was then performed using the specific upstream primer OaFABP4-sgRNA-F and the specific downstream primer sgRNA-R to obtain target fragment 2. S4. The Lenti-CRISPR V2-Puro vector plasmid was digested with restriction endonucleases EcoRI and KpnI, and the linearized vector Lenti-CRISPR V2-Puro was obtained after gel recovery. S5. Ligate target fragment 1 and target fragment 2 obtained in S3 with the linearized vector Lenti-CRISPR V2-Puro obtained in S4. After extracting the plasmid, obtain the Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid. S6. The Lenti-CRISPR V2-Puro-OaFABP4-sgRNA recombinant plasmid and lentiviral packaging plasmid obtained in S5 were co-transfected into HEK293T cells to obtain lentiviral solution. S7. The lentivirus solution obtained in S6 was used to infect Tan sheep preadipocytes. After drug screening with puromycin hydrochloride solution, a cell line with the FABP4 gene knocked out of Tan sheep was obtained. S8. After digesting the cell line with the knockout of the Tan sheep FABP4 gene obtained in S7 with trypsin, perform single-clone screening to obtain a single-clone cell line with the knockout of the Tan sheep FABP4 gene.

4. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 3, characterized in that, The PCR amplification reaction system described in S3 is as follows: 1 μL of sgRNA-F / OaFABP4-sgRNA-F, 1 μL of OaFABP4-sgRNA-R / sgRNA-R, 1 μL of Lenti-CRISPR V2-Puro vector plasmid, 12.5 μL of 2×Phanta Max Master Mix, and ddH2O to a final volume of 25 μL; the reaction program is as follows: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 30 s, 72℃ for 30 s, for 35 cycles; 72℃ for 5 min.

5. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 3, characterized in that, The double digestion reaction system described in S4 is as follows: 10 μg of Lenti-CRISPR V2-Puro vector plasmid, 10 μL of restriction endonuclease EcoRI, 10 μL of KpnI, 20 μL of 10×M Buffer, and ddH2O to a final volume of 200 μL; the double digestion reaction procedure is as follows: double digestion at 37℃ for 2 h.

6. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 3, characterized in that, The ligation system in S5 was as follows: target fragment 1 25 ng, target fragment 2 25 ng, linearization vector Lenti-CRISPRV2-Puro 145 ng, 2×Seamless Cloning Mix 5 μL, and ddH2O to a final volume of 10 μL. The ligation procedure was as follows: ligation was performed at 50 °C for 45 min.

7. The method for constructing a monoclonal cell line with the FABP4 gene knocked out from Tan sheep according to claim 3, characterized in that, The lentiviral packaging plasmids described in S6 are PSPAX2 plasmid and PMD2.G plasmid; the co-transfection time is 48 h; the infection time in S7 is 48 h; the mass concentration of the puromycin hydrochloride solution in S7 is 2 μg / mL; the mass fraction of the trypsin in S8 is 0.25%.

8. An application of a monoclonal cell line of Tan sheep with the FABP4 gene knocked out by the method described in any one of claims 1-7, characterized in that, The application of the monoclonal cell line with the knockout of the FABP4 gene in Tan sheep in constructing a cell model of the regulatory mechanism of Tan sheep precursor adipocyte differentiation.

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