Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage
As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and oth...
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MDPI AG
2022-04-01
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| Online Access: | https://www.mdpi.com/2075-1729/12/5/630 |
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| author | Jing Wang Sami Ullah Khan Pan Cao Xi Chen Fengchong Wang Di Zou Honghui Li Heng Zhao Kaixiang Xu Deling Jiao Chang Yang Feiyan Zhu Yaxuan Zhang Yanhua Su Wenmin Cheng Baoyu Jia Yubo Qing Muhammad Ameen Jamal Hong-Ye Zhao Hong-Jiang Wei |
| author_facet | Jing Wang Sami Ullah Khan Pan Cao Xi Chen Fengchong Wang Di Zou Honghui Li Heng Zhao Kaixiang Xu Deling Jiao Chang Yang Feiyan Zhu Yaxuan Zhang Yanhua Su Wenmin Cheng Baoyu Jia Yubo Qing Muhammad Ameen Jamal Hong-Ye Zhao Hong-Jiang Wei |
| author_sort | Jing Wang |
| collection | DOAJ |
| description | As a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from <i>Diannan</i> miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-β1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases. |
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| spelling | doaj.art-80f7bc58455c40b2810a3f91833ed7272023-11-23T11:49:03ZengMDPI AGLife2075-17292022-04-0112563010.3390/life12050630Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver DamageJing Wang0Sami Ullah Khan1Pan Cao2Xi Chen3Fengchong Wang4Di Zou5Honghui Li6Heng Zhao7Kaixiang Xu8Deling Jiao9Chang Yang10Feiyan Zhu11Yaxuan Zhang12Yanhua Su13Wenmin Cheng14Baoyu Jia15Yubo Qing16Muhammad Ameen Jamal17Hong-Ye Zhao18Hong-Jiang Wei19Key Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaKey Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, ChinaAs a member of the PIKs family, PIK3C3 participates in autophagy and plays a central role in liver function. Several studies demonstrated that the complete suppression of PIK3C3 in mammals can cause hepatomegaly and hepatosteatosis. However, the function of PIK3C3 overexpression on the liver and other organs is still unknown. In this study, we successfully generated PIK3C3 transgenic pigs through somatic cell nuclear transfer (SCNT) by designing a specific vector for the overexpression of PIK3C3. Plasmid identification was performed through enzyme digestion and transfected into the fetal fibroblasts derived from <i>Diannan</i> miniature pigs. After 2 weeks of culturing, six positive colonies obtained from a total of 14 cell colonies were identified through PCR. One positive cell line was selected as the donor cell line for SCNT for the construction of PIK3C3transgenic pigs. Thirty single blastocysts were collected and identified as PIK3C3 transgenic-positive blastocysts. Two surrogates became pregnant after transferring the reconstructed embryos into four surrogates. Fetal fibroblasts of PIK3C3-positive fetuses identified through PCR were used as donor cells for SCNT to generate PIK3C3 transgenic pigs. To further explore the function of PIK3C3 overexpression, genotyping and phenotyping of the fetuses and piglets obtained were performed by PCR, immunohistochemical, HE, and apoptosis staining. The results showed that inflammatory infiltration and vacuolar formation in hepatocytes and apoptotic cells, and the mRNA expression of NF-κB, TGF-β1, TLR4, TNF-α, and IL-6 significantly increased in the livers of PIK3C3 transgenic pigs when compared with wild-type (WT) pigs. Immunofluorescence staining showed that LC3B and LAMP-1-positive cells increased in the livers of PIK3C3 transgenic pigs. In the EBSS-induced autophagy of the porcine fibroblast cells (PFCs), the accumulated LC3II protein was cleared faster in PIK3C3 transgenic (PFCs) thanWT (PFCs). In conclusion, PIK3C3 overexpression promoted autophagy in the liver and associated molecular mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR, causing liver damage in pigs. Therefore, the construction of PIK3C3 transgenic pigs may provide a new experimental animal resource for liver diseases.https://www.mdpi.com/2075-1729/12/5/630PIK3C3transgenic pigsliver damageautophagy |
| spellingShingle | Jing Wang Sami Ullah Khan Pan Cao Xi Chen Fengchong Wang Di Zou Honghui Li Heng Zhao Kaixiang Xu Deling Jiao Chang Yang Feiyan Zhu Yaxuan Zhang Yanhua Su Wenmin Cheng Baoyu Jia Yubo Qing Muhammad Ameen Jamal Hong-Ye Zhao Hong-Jiang Wei Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage Life PIK3C3 transgenic pigs liver damage autophagy |
| title | Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage |
| title_full | Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage |
| title_fullStr | Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage |
| title_full_unstemmed | Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage |
| title_short | Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage |
| title_sort | construction of pik3c3 transgenic pig and its pathogenesis of liver damage |
| topic | PIK3C3 transgenic pigs liver damage autophagy |
| url | https://www.mdpi.com/2075-1729/12/5/630 |
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