Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

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

doi:10.3390/life12050630

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

Life (Basel). 2022 Apr 24;12(5):630. doi: 10.3390/life12050630.

Authors

Jing Wang^{1

2

3}, Sami Ullah Khan^{1

2

3}, Pan Cao^{1

2

4}, Xi Chen^{1

2

3}, Fengchong Wang^{1

2

5}, Di Zou^{1

2

4}, Honghui Li^{1

2

3}, Heng Zhao^{1

2

5}, Kaixiang Xu^{1

2

3}, Deling Jiao^{1

2

3}, Chang Yang^{1

2

5}, Feiyan Zhu^{1

2

5}, Yaxuan Zhang^{1

2

5}, Yanhua Su^{1

2

5}, Wenmin Cheng^{1

2

3}, Baoyu Jia^{1

2

5}, Yubo Qing^{1

2

5}, Muhammad Ameen Jamal^{1

2

3}, Hong-Ye Zhao^{1

2

4}, Hong-Jiang Wei^{1

2

3

4}

Affiliations

¹ Key Laboratory for Porcine Gene Editing and Xenotransplantation in Yunnan Province, Kunming 650201, China.
² Xenotransplantation Research Engineering Center in Yunnan Province, Yunnan Agricultural University, Kunming 650201, China.
³ Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
⁴ State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, China.
⁵ College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China.

Abstract

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 Diannan 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.

Keywords: PIK3C3; autophagy; liver damage; transgenic pigs.

Abstract

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