In vitro development of porcine transgenic nuclear-transferred embryos derived from newborn Guangxi Bama mini-pig kidney fibroblasts

Abstract

Porcine transgenic cloning has potential applications for improving production traits and for biomedical research purposes. To produce a transgenic clone, kidney fibroblasts from a newborn Guangxi Bama mini-pig were isolated, cultured, and then transfected with red and green fluorescent protein genes using lipofectamine for nuclear transfer. The results of the present study show that the kidney fibroblasts exhibited excellent proliferative capacity and clone-like morphology, and were adequate for generation of somatic cell nuclear transfer (SCNT)-derived embryos, which was confirmed by their cleavage activity and blastocyst formation rate of 70.3% and 7.9%, respectively. Cells transfected with red fluorescent protein genes could be passed more than 35 times. Transgenic embryos cloned with fluorescent or blind enucleation methods were not significantly different with respect to cleavage rates (92.5% vs. 86.8%, p > 0.05) and blastocyst-morula rates (26.9% vs. 34.0%, p > 0.05), but were significantly different with respect to blastocyst rates (3.0% vs. 13.2%, p < 0.05). Cleavage (75.3%, 78.5% vs. 78.0%, p > 0.05), blastocyst (14.1%, 16.1% vs. 23.1%, p > 0.05) and morula/blastocyst rates (43.5%, 47.0% vs. 57.6%, p > 0.05) were not significantly different between the groups of transgenic cloned embryos, cloned embryos, and parthenogenetic embryos. This indicates that long-time screening by G418 caused no significant damage to kidney fibroblasts. Thus, kidney fibroblasts represent a promising new source for transgenic SCNT, and this work lays the foundation for the production of genetically transformed cloned Guangxi Bama mini-pigs.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration

“…SCNT was performed as described previously (65)(66)(67)(68). Briefly, gene-edited fibroblasts were thawed and cultured in serum starvation conditions (DMEM/F12 supplemented with 0.5% FBS) for 2 days to synchronize the cell cycle.…”

Production of hGFAP-DsRed transgenic Guangxi Bama mini-pigs via somatic cell nuclear transfer

“…The procedures for ovary collection and in vitro maturation of oocytes were as described previously (Liu et al, 2014) Follicular fluid and cumulus-oocyte complexes (COCs) were aspirated from follicles 3-6 mm in diameter Oocytes with uniform cytoplasm and at least three layers of intact cumulus cells were selected and washed three times in TL-HEPES containing 0.1% w/v polyvinyl alcohol (PVA), and washed twice with maturation medium TCM199 (Invitrogen). Thirty COCs were cultured in each 200 μL TCM-199 medium at 39°C and 5% CO 2 in air with 100% humidity.…”

“…SCNT was performed as described previously (Liu et al, 2014) Briefly, after maturation culture for 42-46 h, oocytes with a first polar body and a uniform cytoplasm were selected for nuclear transfer. The first polar body and the metaphase II spindle plate with a small amount of surrounding cytoplasm were removed and a single donor cell with a smooth surface was transferred into the perivitelline space through the same hole.…”

“…Due to the many physiological, anatomical, and genetic similarities between pigs and humans, there is considerable interest in their use in biomedical research. Miniature pigs such as Bama minipigs are of particular interest because their small size is an advantage for studies in biomedicine, human metabolism, pharmacology, and vascular imaging (Li et al, 2006;Mei et al, 2010;Liu et al, 2008Liu et al, , 2014.…”

Molecular cloning, sequence characteristics, and tissue expression analysis of the SLC35D3 gene in lean, obese and mini-type pigs

“…), dermal regeneration [10], pharmacology, and toxicological studies [11][12], and vascular imaging [13]. It is also useful in the production of food for the elderly, because it fully meet the needs of human physiology and health [6][7][8]. Obesity is a complex disease influenced by genetic and environmental factors and their interactions, phenotypic difference is mainly determined by genetic differences [14][15].…”