| Pig pluripotent stem cells has important significance and application value in regenerative medicine and economy development.At present, several pig induced pluripotent stem(iPS) cell lines have been established by seven groups, but only one of them reported their pig iPS cells contributed to chimera. Up to now, all the pig iPS cell lines are inefficient in exogenous gene silencing, which marked for reprogramming degree. One of reasons for the incomplete reprogramming might be the cross-species origin of induced factor, which come from mouse or human. Different structure of exogenous protein reduced the affinity between induced factor and their enogenerous target genes and interacted proteins, which must be activated for reestablishing pluripotency-gerverment network in the complete reprogramming. In addition, the induce factor group(Oct4,, Sox2, K1f4, c-Myc) suitable for mouse and human might not be sufficient and other fator are needed complete reprogramming in pig induced pluriptent stem cells. In order for optimization of induction systerm of pig induced pluripotent stem cells and better understanding of reprogramming mechanism, current work current study try to get several enogenerous pluripotency genes function in reprogramming and pluripotency maintainace and analysis the work mechanism reprogramming be overexpression in pig flbroblasts. The research content and main results were as follows:(1) In this study, we obtained a variety of porcine pluripotency genes, including Oct4, Sox2, Klf4, c-Myc, Nanog, Eras, and Tell. Sequence analysis proved that the cloned genes well matched with sequence submitted in NCBI and Ensemble databases with more than99%identity in DNA sequences and100%identity in protein except for one amino acid mutation in Nanog.(2) We first cloned porcine Nr5a2gene full-length cDNA by RT-PCR and RACE and obtained two transcript variants of Nr5a2gene.(3) Protein sequence alignment among species proved that protein sequences of all the cloned genes are hightest homology with cloven-hoofed animals, while contrast with rodents and primates the homology decreased.The phylogenetic analysis verified similar results in the evolution.(4) Domain analysis shows that differentiated protein sequences in functional sites of domain from pig, mouse, and human pluripotency protein.(5) We successfully constructed the porcine Oct4, Sox2, K1f4of c-Myc, Nanog gene retroviral vector.After packaged and concentrated, the retrovira were infected porcine flbroblasts. The Immunofluorescence results showed a closed100%infection efficiency(6) Fibroblasts of Oct4overexpressed show20times higher gene expression of Oct4than control. Result of western Blot verified the high expression of Oct4protein.(7) Oct4gene over-expression in fibroblasts activated a variety of pluripotency genes with a3fold increased of Sox2, c-Myc and Tcl1. a4-6fold increased of Klf4and Nanog,and a7-10fold increased of lin28and utf1. Immunofluorescence test of lin28and sall4verified the two protein expression(8) Significantly improvement has been shown in the Oct4over-expressed fibroblast in proliferation ability with a shortened doubling time.(9) Oct4genes take part in regulating cell cycle-related gene expression, five genes including cdc25a,cdk2,ccne1ccnb2,ccnf that funtion in Gap-crossing of G1and G2showed up-regulated.(10) Oct4gene over-expression involved in the up-regulation of epigenetic modification-related genes aride1a, jarid1b,setdb1,dnmt3b.(11) Oct4overexpressed fibroblasts could form a number of stem cell-like clones.(12) Oct4overexpression increased tight junction-related genes including cldn11, ocln, cldn3. |
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