The Study Of Highly Efficient Generation Of Sheep Induced Pluripotent Stem Cell

The sheep has long been an important economic animal. Transgenic technology in sheep can quickly generate new sheep varieties with high quality and high yield. However, traditional transgenic techniques result in the extremely low efficiency and developmental disorder. The gene targeting technique based on embryonic stem cells(ESCs) can greatly improve the efficiency of gene modified mouse, nevertheless, despite intensive efforts, derivation of ESCs form sheep remains elusive. As an alternative pluripotent cell type to sheep ESC, sheep induced pluripotent stem cells(si PSCs), which derived from sheep somatic cells by transduction defined factors, would able to be used in generating transgenic animals. Although several groups obtained the si PSCs, the reprogramming effiency still extremely low. Therefore, explore a highly efficiency way to generate si PSCs of the future are a top priority. In the future, si PSCs may not only be used to generate gene modified sheep, but aslo study the mechanisms of reprogramming and development. These cells also hold the great hope in directed differentiation into functional cells such as germ cells.Objective:(1) Separated three sheep somatic cells in order to explore the best start candidate cell for inducing si PSCs, preparation of feeder cells for cultivating the si PSCs;(2) Constructed p53 interfering lentivirus plasmid in order to promote the efficiency of reprogramming;(3) Constructed ASF1 A overexpression plasmid to improve the efficiency of reprogramming;(4) Highly efficient generation of si PSCs.Methods:(1) The sheep primary fibroblasts, kidney cells and bone marrow mesencheymal stem cells were derived from a 45 d Chinese Merino fetal sheep by tissue culture and whole bone marrow adherence method, respectively. The mouse embryonic fibroblasts were derivied from CF-1 mice fetals at 12.5 days, X- ray irradiated the P3 MEFs to preparate feeder cells;(2) According to the sheep p53 gene sequences in Gen Bank database, design of interference of p53 short hairpin RNA(sh RNA) to constructe p LL3.7-p53–sh RNA lentiviral plasmid. Plasmids p LL3.7-p53-sh RNA and p LL3.7 empty carrier with helper plasmids(VSVG, p MDL and REV) were co-transfected into HEK-293 FT cells for packaging of the lentivirus using Ca PO4 precipitation, respectively. Titration of lentiviral vectors and infected SKC with MOI=10. Rreal-time PCR and Western blot were used to identify the interfere efficiency at the m RNA and protein level of the p53 and p21.(3) According to the EGFP and ASF1 A sequences in Genebank database, design primers respectively and amplification them. The c-DNA for EGFP and ASF1 A were cloned into the PLEX-MCs lentiviral vector backbone to construct PLEX-ASF1A-EGFP plasmid. To produce lentiviral particles, HEK293 FT cells were co-transfected with PLEX-ASF1A-EGFP and helper plasmids. SKCs were infected with lentivirus which overexpression ASF1 A. Rreal-time PCR and Western blot were used to identify the m RNA and protein of the ASF1 A.(4) SEF, SKC and BMSC were co-infected with eight defined factors(Oct4/Sox2/c-Myc/Klf4/Lin28/Nanog/Sv40LT/h TERT) described by Bao Lei et al, Alkaline phosphatase(AP) staining was performed on day 23 and to compare the reprogramming efficiency; Additional, chosen the highest efficient of the three sheep somatic cells to obtain the si PSCs with the help of p53-si RNA and ASF1 A, compared their reprogramming efficiency. AP staining, real time PCR, immunostaining, bisulphite genomic sequencing, karyotype analysis, embryoid body formation and teratoma formation were carried out to characterization of reprogrammed si PSCs.Results:(1) We successfully obtained SEFs, SKCs, BMSCs from a fetal sheep and the sheep BMSCs were highly expressed CD44; we also successful preparation of CF-1 feeder cells;(2) We successfully constructed 4 sheep p LL3.7-p53–sh RNA lentiviral plasmids and found that the p53-sh RNA-3 was the most efficient of the four sh RNAs; We observed a significant reduction in the expression of p53 and p21;(3) The c DNA encoding sheep ASF1 A was subcloned into the PLEX-EGFP vector successfully. We forced expression ASF1 A in SKC and found a high ASF1 A expression levels.(4) 8 factors could successfully obtained si PSCs from three sheep somatic cells, the SCKs was the best candidate for inducing si PSCs. P53 reduction and ASF1 A increase could significantly increase the formation of si PSC colonies.Conclusion:(1) We successfully isolated and cultivated three sheep somatic cells, all these cells would provide the materials for the subsequent experimental; we also successfully obtained the feeder layer cells for the cultivation of si PSCs.(2) p53-sh RNA-3 was the best sh RNA for interfering sheep p53, and can significantly reduce the expression of p53 and p21 in SKCs.(3) Forced expression ASF1 A in SKCs could significantly increase the expression of ASF1 A.(4) Compared with SEF and BMSC, SKC was more easily to reprogram into si PSCs. Reduced the expression of p53 and increased the expression of ASF1 A could improve the efficiency of sheep somatic cells reprogramming.