Application of PINK1 gene in porcine trophoblast cells and method for cell line construction
By constructing a porcine trophoblast cell line with PINK1 gene induction and knockout, the proliferation, differentiation, and autophagy of porcine placental trophoblast cells were regulated, thus solving the problem of insufficient placental function in porcines and improving the reproductive performance of sows.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI JIAOTONG UNIV
- Filing Date
- 2026-03-20
- Publication Date
- 2026-06-02
AI Technical Summary
In the prior art, the disordered proliferation and differentiation of porcine placental trophoblast cells leads to placental insufficiency, affecting the reproductive performance of sows, and the regulatory mechanism of PINK1 in porcine placental trophoblast cells has not been reported.
We constructed porcine ectodermal cell lines with PINK1 gene induction and knockout, and achieved precise regulation of PINK1 gene expression by modulating the Parkin-P62-LC3B pathway, thereby activating or inhibiting the expression of related genes.
It promotes cell proliferation, inhibits apoptosis, increases autophagy levels, and enhances placental function and efficiency, providing a reliable cell model for studying porcine placental development and disease mechanisms.
Smart Images
Figure CN122128245A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biotechnology and animal reproduction technology, specifically relating to the application of the PINK1 gene in porcine trophic ectoderm cells and the method for constructing cell lines. Background Technology
[0002] China is the world's largest producer of pigs, accounting for 48% of global pig production in 2024 / 2025. However, the "2024-2029 China Pig Farming and Slaughtering Industry Market Panorama Survey and Development Prospect Forecast Report" shows that in 2024, my country's sow PSY (Pigs per Sow per Year) was only 25.05 heads, still significantly lower than countries like Denmark. Low sow productivity is a bottleneck restricting the development of my country's pig farming industry. The number of live piglets per litter and the birth weight of piglets directly determine the sow's productivity. Uteroplacental efficiency is a crucial determinant of embryonic survival and development in early pregnancy; insufficient placental efficiency leads to intrauterine growth retardation (IUGR) and increased embryonic mortality, seriously affecting the healthy development of the pig industry.
[0003] Placental efficiency is influenced by the development of placental folds and blood vessels, and the proliferation and invasion of placental trophoblast cells affect the formation of placental folds. The proliferation and differentiation of placental trophoblast cells effectively enhance placental function and efficiency, i.e., the placenta's ability to deliver nutrients to the fetus. Disorders in trophoblast cell proliferation and differentiation may be associated with abnormal placental formation, leading to various pregnancy complications such as miscarriage, stillbirth, and fetal growth restriction. Studies have shown that during pregnancy, trophoblast cells at the top and base of placental folds differentiate into two cell types, and changes in trophoblast cell morphology have a crucial impact on porcine placental development. In early embryonic development, the placental chorion is not tightly adhered to the uterus. As pregnancy progresses, the thickness of the placental matrix layer increases significantly, and blood vessels increase. In mid-pregnancy, the placental trophoblast epithelium and the endometrial cavity epithelium form placental folds through cell migration and remodeling; the trophoblast epithelial cells have strong proliferative capacity and are surrounded by dense microvessels. In late pregnancy, the thickness of the placental matrix layer gradually decreases, and the placental folds further develop. In this process, Cdx2 not only maintains the function of trophoblast cells but also participates in the formation and differentiation of placental villi, ensuring the normal development of placental structure and playing a crucial role in embryo implantation and placental formation. Studies have shown that the presence of Maspin is a prerequisite for successful embryo implantation; it can prevent excessive invasion of maternal uterine tissue by limiting the excessive invasion of extratrophoblast cells, thereby maintaining the normal structure of the placenta. The CTBP2 gene participates in regulating the expression of genes related to placental angiogenesis and in regulating the proliferation and differentiation of hematopoietic stem cells, which is crucial for the development of the placental vascular system and the establishment of placental function.
[0004] The PINK1 / Parkin signaling pathway is one of the most important pathways of mitophagy. When mitochondria undergo depolarization, damage, or dysfunction, PINK1 protein expression increases, accumulating on the damaged outer mitochondrial membrane and recruiting Parkin ubiquitinated proteins. Parkin, a phosphorylated physiological substrate of PINK1, possesses E3 ubiquitin-protein ligase activity and mediates the ubiquitination of substrate proteins on the outer mitochondrial membrane. This allows it to be recognized by the autophagy receptor protein p62, which then binds to LC3B, forming a mitophagosome. This process degrades damaged mitochondria and maintains cellular homeostasis. LC3B and p62 are important marker proteins in autophagy. During autophagy, LC3B in the autophagosome transforms from cytoplasmic LC3B-I to membrane-bound LC3B-II. p62, an autophagy substrate, selectively enters the autophagosome and is degraded after binding to LC3B and ubiquitinated proteins. Currently, there are no reports on the role and mechanism of PINK1 in regulating the proliferation, differentiation, and apoptosis of porcine placental trophoblast cells. Therefore, by constructing porcine placental trophoblast ectoderm cell lines with PINK1-induced expression and knockout, we aim to achieve precise regulation of PINK1 expression levels and deeply analyze the role and mechanism of PINK1 in regulating autophagy, proliferation, differentiation, and apoptosis of porcine placental cells, providing a reference method for the subsequent establishment of porcine PINK1-related cell lines. Summary of the Invention
[0005] The technical problem to be solved: The purpose of this invention is to provide the application of the PINK1 gene in porcine trophoblast cells, and a method for constructing PINK1 gene-induced expression and knockout cell lines. Through the expression of related genes and proteins in successfully constructed cell lines, the effects of PINK1 gene expression levels on the proliferation, differentiation, and autophagy of porcine trophoblast cells, as well as placental function and efficiency, were confirmed. The results show that induced expression of PINK1 promotes cell proliferation and inhibits apoptosis; this provides a reliable basis for the regulatory mechanism of oxidative stress and placental development in porcine placental trophoblast cells, and provides a new technical means to improve sow reproductive performance.
[0006] Technical solution: Application of PINK1 gene in porcine trophoblast cells, wherein the expression of PINK1 gene can regulate the proliferation, differentiation and autophagy of porcine trophoblast cells, and improve placental function and efficiency.
[0007] Furthermore, the expression of the PINK1 gene includes inducing expression of the PINK1 gene or knocking out the PINK1 gene.
[0008] Furthermore, the nucleotide sequence of the induced expression of the PINK1 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the knockout of the PINK1 gene is shown in SEQ ID NO.2.
[0009] Furthermore, the induced expression of the PINK1 gene can activate the Parkin-P62-LC3B pathway and increase the expression of Mgst1, CTBP2 and IGFBP3 genes.
[0010] Furthermore, the knockout of the PINK1 gene can inhibit the Parkin-P62-LC3B pathway, reduce the expression of Mgst1, CTBP2 and IGFBP3 genes, and increase the expression of Cdx2.
[0011] A method for constructing a PINK1 gene-induced expression porcine trophic ectoderm cell line, comprising the following steps: S1. Construct an inducible expression vector containing the PINK1 gene: the vector element sequence is TetIIP-MCS-3FLAG-Ubi-TetR-IRES-Puromycin; S2. Lentiviral packaging: The inducible expression vector and packaging plasmids PMD2.G and PAX2 were co-transfected into 293T cells. Cell supernatant was collected, filtered and sterilized to obtain lentivirus. S3. Cell line construction: Porcine ectoderm cells were infected with lentivirus, polybrene was added to promote infection, and puromycin was added for screening. Single clones were selected to verify the induction expression effect and construct the PINK1 inducible expression cell line.
[0012] The PINK1 gene-inducible porcine ectoderm cell line constructed by the above method is capable of PINK1 gene expression induced by doxycycline.
[0013] A method for constructing a PINK1 gene knockout porcine trophoblast cell line includes the following steps: S1. Construction of PINK1 gene knockout plasmid: gRNA was designed targeting the exons of the porcine PINK1 gene, and the gRNA was ligated into the digested Lenti-CRISPR-V2 vector using a ligase to construct the PINK1 gene knockout plasmid. S2. Lentiviral packaging: PINK1 gene knockout plasmid and packaging plasmids PMD2.G and PAX2 were co-transfected into 293T cells, cell supernatant was collected, filtered and sterilized to obtain lentivirus; S3. Cell line construction: Porcine ectoderm cells were infected with lentivirus, polybrene was added to promote infection, and puromycin was added for selection. Single clones were selected to verify the induction expression effect and construct the PINK1 gene knockout cell line.
[0014] Further, the sequence of the gRNA in step S1 is shown in SEQ ID NO.3-5; the gRNA is ligated to the enzyme-digested Lenti-CRISPR-V2 vector by: digesting the Lenti-CRISPR-V2 vector with BsmBI enzyme, performing agarose gel electrophoresis on the digestion products, recovering large fragment digestion products from the gel, and then ligating the gRNA to the digestion products with T4 ligase.
[0015] The PINK1 gene-induced expression porcine trophoblast cell lines and PINK1 gene knockout porcine trophoblast cell lines constructed by the methods described above can be used to study porcine placental development, the mechanisms of diseases related to placental dysfunction, or to screen drugs that regulate placental function. Beneficial effects
[0016] This invention discovers that the PINK1 gene affects autophagy, proliferation, and differentiation of porcine trophoblast cells by regulating the Parkin-P62-LC3B pathway, providing a new theoretical basis for understanding the molecular mechanisms of porcine placental development. Furthermore, it successfully constructed porcine trophoblast cell lines with PINK1-induced expression and knockout, providing a reliable cell model for studying the mechanisms of porcine placental development and placental dysfunction-related diseases.
[0017] This invention discovers that induced expression of PINK1 can promote cell proliferation, inhibit apoptosis, and increase autophagy levels. Furthermore, it enhances placental function and efficiency by upregulating the expression of Mgst1, CTBP2, and IGFBP3 genes. The Tet-On induction system allows for precise regulation of PINK1 gene expression. By controlling doxycycline concentration and induction time, the effects of different PINK1 expression levels on cell function can be studied.
[0018] The technical solution of this invention can be applied to improve sow reproductive performance, improve placental efficiency, increase the number of live piglets per litter and the birth weight of piglets, which is of great significance to the development of the pig industry. Attached image description: Figure 1 Figure showing the construction and validation results of a monoclonal cell line induced to express the PINK1 gene; Figure 2 Figure showing the verification results of cell line construction with PINK1 gene knockout; Figure 3 The effect of induced PINK1 gene expression on cell viability is shown in the figure. Figure 4 Figure showing the effect of PINK1 gene induction on the expression of autophagy, proliferation, and apoptosis proteins; Figure 5 Figure showing the results of gene level verification in cells induced to express the PINK1 gene; Figure 6Figure showing the effect of PINK1 gene knockout on the expression of autophagy, proliferation, and apoptosis proteins; Figure 7 The graph shows the expression results of related genes in cell lines with the PINK1 gene knocked out. Detailed Implementation
[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1
[0020] Cell culture: Porcine ectoderm cells (pTr) were cultured in DMEM / F12 medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. Culture conditions were a 37°C, 5% CO2 saturated humidity incubator. Cell passage was performed using 0.25% trypsin digestion. Example 2
[0021] Construction of the PINK1 gene-induced expression vector: The porcine gene PINK1 (SEQ ID NO. 6) was selected. The vector element sequence was: TetIIP-MCS-3FLAG-Ubi-TetR-IRES-Puromycin, and the vector size was 12362 bp. The vector contains the TetIIP promoter (2713-2991), 3FLAG (3028-3105), and TetR (4372-5073), and can be used for 3×FLAG-PINK1 fusion protein expression. Vector map reference: http: / / www.genechem.com.cn / service / index.php?ac=gene&at=vector_search&keyword=GV308 The inserted fragment is located in the MCS, and the primer sequence for the target gene PINK1(97601-1)-p1 is as shown in SEQ ID NO.7. Example 3
[0022] PINK1 gene knockout plasmid construction: Three PINK1 gRNAs were designed from the porcine PINK1 gene exons, with sequences shown in SEQ ID NO. 3-5. After primer annealing, the Lenti-CRISPR-V2 template was digested with BsmBI, and the band size was verified by gel extraction. The plasmids were ligated with T4 ligase, transformed into competent E. coli cells, plated, incubated overnight at 37°C, and single clones were selected for sequencing. The successfully constructed plasmids were then expanded and cultured for extraction. Example 4
[0023] Lentiviral packaging: 293T cells were packaged at a ratio of 2×102 6Seeds were placed in 10cm cell culture dishes and cultured overnight. Two sterile centrifuge tubes, A and B, were used. In tube A, 5μg of the target plasmid, 1.8μg of the packaging plasmid PMD2.G, and 3.2μg of the PAX2 plasmid were added, along with 500μL of OPTI-MEM. The mixture was gently pipetted and incubated for 5 min. Then, in tube B, 20μL of transfection reagent (EZ Trans cell transfection reagent) and 500μL of OPTI-MEM were added and gently pipetted and mixed. The liquid from tube A was then added to tube B, mixed, and incubated at room temperature for 15 min. The above liquid was then added to 293T cells, and the cells were cultured for another 48 h. The cell supernatant was obtained by centrifugation and filtered through a 0.45μm filter. Example 5
[0024] Construction of PINK1 gene-induced expression cell lines: pTr cells were constructed at a rate of 2 × 10⁻⁶. 5 Cells were densely seeded in 6-well plates and cultured overnight. 300 μL of viral particles and 8 μg / mL polybrene were added for infection. After 48 h, 2 μg / mL puromycin was added for continuous selection for 3-7 days to obtain monoclonal cells. Approximately 30 monoclonal cells were randomly selected, and Western blot was used to verify the PINK1-induced expression effect.
[0025] Monoclonal cells were treated with 1-10 μg / mL DOX for 48 h, and total protein was extracted. FLAG protein was detected by Western blot. Figure 1 The results showed that FLAG protein expression generally increased with increasing DOX concentration, indicating that the induced expression cell line was successfully constructed. Example 6
[0026] PINK1 gene knockout cell line construction: pTr cells were constructed at a rate of 2 × 10⁻⁶. 5 Cells were densely seeded in 6-well plates and cultured overnight. 300 μL of viral particles and 8 μg / mL polybrene were added for infection. After 48 hours, 2 μg / mL puromycin was added for continuous selection for 3-7 days to obtain monoclonal cells. Approximately 30 monoclonal cells were randomly selected for Western blot verification of PINK1 knockout.
[0027] PINK1 protein was detected using Western blot. Figure 2 The results showed that the PINK1 protein was almost not expressed when the sgRNA-1 and sgRNA-3 target knockouts were used, indicating that the PINK1 knockout cell line was successfully constructed. The sgRNA-1 group was then used for further experiments. Example 7
[0028] Cell line validation Protein expression detection: PINK1-induced and knockout pTr cells were arranged at a density of 1×10⁻⁶.5 Cells were seeded at 10 mL / mL in 100 mm cell culture dishes and allowed to adhere for 24 h. After treatment, the supernatant was removed, and the cells were washed twice with PBS. 1 mL of RIPA protein lysis buffer (KGP703) containing a protease inhibitor (Roche 05892791001) was added to each dish. Cells were scraped from the wells using a cell scraper, transferred to centrifuge tubes, and lysed on ice for 30 min. The cells were then centrifuged at 12000 g for 30 min, and the supernatant was collected. The antibodies used in the experiment are shown in Table 1 below.
[0029] Table 1 Antibody source and dilution ratio
[0030] Gene expression detection: After the cells were constructed, they were washed twice with PBS, and 700 μL of TRK lysis buffer was added. RNA was extracted from the cells using an RNA extraction kit (R6841-01, Omega). The concentration of extracted RNA was measured using a NanoDrop-100. An A260 / 280 ratio between 1.8 and 2.0 indicated good RNA purity. The RNA was then reverse transcribed into cDNA using a reverse transcription kit (R323-01, Nanjing Novizan Biotechnology Co., Ltd.).
[0031] The primer sequences are shown in Table 2 below. They were synthesized using Primer 3 Input software and were synthesized by Invitrogen Shanghai Co., Ltd.
[0032] Table 2 Primer Sequences
[0033] The quantitative PCR reaction system is shown in Table 3. Each sample was replicated in triplicate. The reaction conditions were as follows: Step 1: 95℃ for 30s; Step 2: 95℃ for 5s, 60℃ for 34s, repeated 40 times; Step 3: 95℃ for 15s, 60℃ for 60s, 95℃ for 15s. The quantitative PCR results were obtained using the 2-(ΔΔCt) method, where ΔΔCt = (Ct Target - Ct β-actin) treatment group - (Ct Target - Ct β-actin) control group, and the relative expression levels of the genes in the test samples were calculated.
[0034] Table 3 Quantitative PCR System
[0035] Example 8 The effect of induced PINK1 gene expression on cell viability: The effect of 10 μg / mL DOX treatment for different durations on cell viability was determined using a successfully constructed induced expression cell line.
[0036] Figure 3 The results showed that after 24h and 48h of treatment, the cell viability of the 10μg / mL DOX group was significantly lower than that of the control group, while there was no significant change in cell viability at 6h and 12h.
[0037] Effects of induced PINK1 gene expression on autophagy, proliferation, and apoptosis proteins and gene expression: Cell lines with successful induced expression were treated with 0 and 10 μg / mL DOX for 12 h and 0 and 0.3 mM H2O2 for 12 h, respectively. Western blot was used to detect the expression levels of Flag, PINK1, Parkin, p62, LC3B, PCNA, and CytC proteins, and quantitative PCR was used to detect the expression of the target gene.
[0038] Depend on Figure 4 The results showed that under normal conditions and H2O2 treatment, induced expression of PINK1 significantly reduced the protein expression levels of Parkin and p62, while slightly increasing the expression level of LC3B. P62 expression was negatively correlated with autophagy, while LC3B expression was positively correlated with autophagy. This indicates that induced expression of PINK1 increased autophagy levels, particularly mitophagy. The increased PCNA protein expression and decreased CytC protein expression suggest that induced expression of PINK1 promotes cell proliferation and inhibits apoptosis.
[0039] Effects of induced PINK1 gene expression on proliferation, differentiation, gene expression, and gene expression: Figure 5 The results showed that the gene expression of Cdx2 and IGFBP3 did not differ under different DOX concentrations. However, the gene expression of Maspin, Mgst1, and CTBP2 increased significantly under 10 μg / mL DOX treatment, indicating that induced expression of PINK1 promoted the expression of Maspin, Mgst1, and CTBP2 genes. Example 9
[0040] Effects of PINK1 gene knockout on autophagy, proliferation, and apoptosis proteins: Normal and PINK1-knockout placental trophoblast cells were treated with 0.3 mM H2O2 for 12 h. Western blot was used to detect the protein expression levels of PINK1, Parkin, p62, and LC3B. Quantitative PCR was used to detect gene expression.
[0041] Figure 6The results showed that knocking out PINK1 significantly reduced the expression levels of Parkin, P62, and PCNA proteins, while LC3B expression increased under normal conditions. However, under H2O2 treatment, the expression levels of Parkin, P62, LC3B, and PCNA decreased, while the expression level of CytC increased, indicating that under stress conditions, knocking out PINK1 reduced autophagy and proliferation, and increased apoptosis.
[0042] like Figure 7 After knocking out the PINK1 gene, under normal conditions, Maspin expression showed no significant difference, while Cdx2 expression increased, and the expression levels of Mgst1, CTBP2, and IGFBP3 decreased significantly. Under H2O2 treatment, the expression trends of Cdx2, Mgst1, CTBP2, and IGFBP3 were the same as under normal conditions, while Maspin expression increased. Therefore, knocking out PINK1 reduces the expression of IGFBP3, Mgst1, and CTBP2, while increasing the expression of Cdx2.
[0043] In summary, this invention successfully constructed a stable porcine placental trophoblast cell line capable of inducible expression and PINK1 knockout. PINK1-induced expression activates the Parkin-P62-LC3B pathway, promotes mitophagy, and increases the expression of Mgst1 and CTBP2 genes; conversely, PINK1 knockout inhibits the Parkin-P62-LC3B pathway, reduces autophagy levels, and decreases the expression of Mgst1, CTBP2, and IGFBP3 genes. Mgst1 participates in intracellular antioxidant stress responses, helping to maintain intracellular redox balance, protecting placental cells from oxidative damage, and ensuring normal placental proliferation and differentiation. CTBP2, as a transcriptional co-regulator, regulates the expression of cyclins and kinases, controls cell entry into different cell cycle stages, modulates the sensitivity of apoptosis-related genes such as Bcl-2, and participates in cell proliferation, differentiation, and apoptosis during placental development. IGFBP3 regulates insulin-like growth factor activity in placental tissue, thus regulating cell proliferation rate. Therefore, PINK1 promotes cell redox balance, proliferation, and differentiation by promoting the expression of Mgst1, CTBP2, and IGFBP3 genes in porcine placental trophoblast cells. Thus, by regulating the expression level of the PINK1 gene, the function and efficiency of porcine placenta can be improved. It can be applied to study the disease mechanism of porcine placental development and placental dysfunction, or to screen drugs for regulating placental function.
[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.
Claims
1. The application of the PINK1 gene in porcine trophoblast cells, characterized by: The expression of the PINK1 gene can regulate the proliferation, differentiation, and autophagy of porcine trophoblast cells, thereby improving placental function and efficiency.
2. The application of the PINK1 gene in porcine trophoblast cells according to claim 1, characterized in that: The expression of the PINK1 gene includes induced expression of the PINK1 gene or knockout of the PINK1 gene.
3. The application of the PINK1 gene in porcine trophoblast cells according to claim 2, characterized in that: The nucleotide sequence of the induced expression of the PINK1 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the knockout of the PINK1 gene is shown in SEQ ID NO.
2.
4. The application of the PINK1 gene in porcine trophoblast cells according to claim 2, characterized in that: The induced expression of the PINK1 gene can activate the Parkin-P62-LC3B pathway and increase the expression of Mgst1, CTBP2 and IGFBP3 genes.
5. The application of the PINK1 gene in porcine trophoblast cells according to claim 2, characterized in that: The knockout of the PINK1 gene can inhibit the Parkin-P62-LC3B pathway, reduce the expression of Mgst1, CTBP2 and IGFBP3 genes, and increase the expression of Cdx2.
6. A method for constructing a PINK1 gene-induced expression porcine trophoblast cell line, characterized in that, Includes the following steps: S1. Construct an inducible expression vector containing the PINK1 gene: the vector element sequence is TetIIP-MCS-3FLAG-Ubi-TetR-IRES-Puromycin; S2. Lentiviral packaging: The inducible expression vector and packaging plasmids PMD2.G and PAX2 were co-transfected into 293T cells. Cell supernatant was collected, filtered and sterilized to obtain lentivirus. S3. Cell line construction: Porcine ectoderm cells were infected with lentivirus, polybrene was added to promote infection, and puromycin was added for screening. Single clones were selected to verify the induction expression effect and construct the PINK1 inducible expression cell line.
7. The PINK1 gene-induced porcine ectodermal cell line constructed according to claim 6, characterized in that: The cell line was able to induce PINK1 gene expression via doxycycline.
8. A method for constructing a PINK1 gene knockout porcine trophoblast cell line, characterized in that, Includes the following steps: S1. Construction of PINK1 gene knockout plasmid: gRNA was designed targeting the exons of the porcine PINK1 gene, and the gRNA was ligated into the enzyme-digested Lenti-CRISPR-V2 vector to construct the PINK1 gene knockout plasmid. S2. Lentiviral packaging: PINK1 gene knockout plasmid and packaging plasmids PMD2.G and PAX2 were co-transfected into 293T cells, cell supernatant was collected, filtered and sterilized to obtain lentivirus; S3. Cell line construction: Porcine ectoderm cells were infected with lentivirus, polybrene was added to promote infection, and puromycin was added for selection. Single clones were selected to verify the induction expression effect and construct the PINK1 gene knockout cell line.
9. The method for constructing a PINK1 gene knockout porcine trophoblast cell line according to claim 8, characterized in that: The sequence of the gRNA in step S1 is shown in SEQ ID NO.3-5; the gRNA is ligated to the digested Lenti-CRISPR-V2 vector by digesting the Lenti-CRISPR-V2 vector with BsmBI enzyme, the digestion products are subjected to agarose gel electrophoresis, the large fragment digestion products are recovered from the gel, and then the gRNA and the digestion products are ligated by T4 ligase.
10. The application of the PINK1 gene-induced expression porcine trophoblast cell lines and PINK1 gene knockout porcine trophoblast cell lines constructed according to any one of claims 6-9 in studying porcine placental development, the mechanism of diseases related to placental dysfunction, or screening drugs for regulating placental function.