Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning

Zhao, Heng; Li, Yuying; Wiriyahdamrong, Thanapa; Yuan, Zaimei; Qing, Yubo; Li, Honghui; Xu, Kaixiang; Guo, Jianxiong; Jia, Baoyu; Zhang, Xuezeng; Cheng, Wood-Hi; Su, Yanhua; Long, Weihu; Wang, Jing; Zou, Di; Kinoshita, Keji; Zhao, Hongyan; Wei, Hong‐Jiang

doi:10.1007/s11248-020-00201-2

Cited by 12 publications

(7 citation statements)

References 31 publications

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“…Fetal fibroblasts of the PIK3C3-positive fetuses were used as donor cells for the second round of SCNT in the generation of PIK3C3-transgenic pigs. According to our previous work, using fibroblast cells derived from fetuses obtained by the first round of cloning can retain the cellular vitality and purify the genotype of gene editing animals [ 24 ]. Hence, we performed the re-cloning process to obtain PIK3C3-transgenic piglets.…”

Section: Resultsmentioning

confidence: 99%

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Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

Wang

Khan

Cao

et al. 2022

Life

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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.

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Section: Resultsmentioning

confidence: 99%

“…Oocyte collection, IVM culturing, and SCNT were performed as described in our previous studies [ 24 , 25 ]. Donor cells from a PIK3C3-positive fibroblast cell line were inserted into the perivitelline space of an enucleated oocyte.…”

Section: Methodsmentioning

confidence: 99%

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

Wang

Khan

Cao

et al. 2022

Life

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“…Highly reprogrammable NDCs may exhibit an enhanced capacity to sustainably perpetuate stemness- and pluripotency-related molecular attributes. The latter, in turn, could enhance the epigenetic dedifferentiation capacity of OE-OCT6 iPSCs in SCNT-derived embryos, conceptuses, and progenies generated for transgenic and biomedical research, not only for pig-to-human xenotransplantation and tissue engineering, but also for regenerative and reconstructive surgical treatments in humans and other mammalian species ( Berthelsen et al, 2021 ; Podstawski et al, 2022 ; Song et al, 2016 ; Wiater et al, 2021a , 2021b ; Xu et al, 2022 ; Yu et al, 2021 ; Zhao et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation

Yang¹,

Wu²,

Li³

et al. 2022

Zoological Research

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As a transcription factor of the Pit-Oct-Unc (POU) domain family, octamer-binding transcription factor 6 ( OCT6 ) participates in various aspects of stem cell development and differentiation. At present, however, its role in porcine-induced pluripotent stem cells (piPSCs) remains unclear. Here, we explored the function of OCT6 in piPSCs. We found that piPSCs overexpressing OCT6 maintained colony morphology and pluripotency under differentiation conditions, with a similar gene expression pattern to that of non-differentiated piPSCs. Functional analysis revealed that OCT6 attenuated the adverse effects of extracellular signal-regulated kinase (ERK) signaling pathway inhibition on piPSC pluripotency by activating phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling activity. Our research sheds new light on the mechanism by which OCT6 promotes PSC maintenance.

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“…(3) sheep [21][22][23][24][25][26]; (4) pigs [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]; (5) equids-domestic horses [42][43][44][45][46] and mules [47]; (6) water buffaloes-Chinese swamp buffaloes [48,49] and Indian river/riverine buffaloes [50][51][52][53]; (7) one-humped or dromedary camels [7,[54][55][56]; (8) two-humped or Bactrian camels [57]; (9) domestic cats [58][59][60][61][62][63]; (10) domestic dogs…”

Section: Biotechnological Possibilities Of Applying the Techniques Ofmentioning

confidence: 99%

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

Samiec

Skrzyszowska

2021

IJMS

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The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

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Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning

Cited by 12 publications

References 31 publications

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

Construction of PIK3C3 Transgenic Pig and Its Pathogenesis of Liver Damage

OCT6 inhibits differentiation of porcine-induced pluripotent stem cells through MAPK and PI3K signaling regulation

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

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