Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

被引：3

作者：

Wang, Jing ^{[1
,2
,3
]}

Khan, Sami Ullah ^{[1
,2
,3
]}

Cao, Pan ^{[1
,2
,4
]}

Chen, Xi ^{[1
,2
,3
]}

Wang, Fengchong ^{[1
,2
,5
]}

Zou, Di ^{[1
,2
,4
]}

Li, Honghui ^{[1
,2
,3
]}

Zhao, Heng ^{[1
,2
,5
]}

Xu, Kaixiang ^{[1
,2
,3
]}

Jiao, Deling ^{[1
,2
,3
]}

Yang, Chang ^{[1
,2
,5
]}

Zhu, Feiyan ^{[1
,2
,5
]}

Zhang, Yaxuan ^{[1
,2
,5
]}

Su, Yanhua ^{[1
,2
,5
]}

Cheng, Wenmin ^{[1
,2
,3
]}

Jia, Baoyu ^{[1
,2
,5
]}

Qing, Yubo ^{[1
,2
,5
]}

Jamal, Muhammad Ameen ^{[1
,2
,3
]}

Zhao, Hong-Ye ^{[1
,2
,4
]}

Wei, Hong-Jiang ^{[1
,2
,3
,4
]}

机构：

[1] Key Lab Porcine Gene Editing & Xenotransplantat Y, Kunming 650201, Yunnan, Peoples R China

[2] Yunnan Agr Univ, Xenotransplantat Res Engn Ctr Yunnan Prov, Kunming 650201, Yunnan, Peoples R China

[3] Yunnan Agr Univ, Fac Anim Sci & Technol, Kunming 650201, Yunnan, Peoples R China

[4] Yunnan Agr Univ, State Key Lab Conservat & Utilizat Bioresources Y, Kunming 650201, Yunnan, Peoples R China

[5] Yunnan Agr Univ, Coll Vet Med, Kunming 650201, Yunnan, Peoples R China

来源：

LIFE-BASEL | 2022年 / 12卷 / 05期

基金：

国家重点研发计划;

关键词：

transgenic pigs; liver damage; autophagy; CARDIAC-HYPERTROPHY; AUTOPHAGY; VPS34; HEART; APOPTOSIS; NECROSIS; STRESS; INJURY; DRAM; PHOSPHORYLATION;

D O I：

10.3390/life12050630

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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-kappa B, TGF-beta 1, TLR4, TNF-alpha, 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.

引用

页数：18

共 62 条

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