Generation Of Induced Pluripotent Stem Cells From Bovine Adult Somatic Cell By Different Transcription Factors

(1) Set of bovine pluripotency genes expression constructs in retroviral pMSCV Vector.Total RNA were isolated from fetal bovine tissue of primordial germ ridge using TRIzol LS reagent and treated with DNA-free reagent to remove DNA contamination. The open reading frames (ORF) sequences of Oct4 and c-Myc genes were amplified by RT-PCR. The PCR products were digested with EcoRI/BgⅢ, and then inserted them into the same restriction sites of retroviral pMSCVnoe vector respectively. All of the fragments were verified by sequencing.(2) Retroviruses generated from PT67 packaging cellsTo monitor the viral infection efficiency in bovine dermal fibroblasts (BDFs), retroviral pMSCVneo-IRES-GFP (MIG) vector was cotransfected with retroviral vector. The single-gene infection efficiency was able to reach about 53.1% and 51.3% respectively in this study. Total RNA was extracted using TRIzol LS reagent. After RT-PCR amplification, the singel bands were acquired. We can observe the virus particles under the transmission electron microscopy. The two populations of NIH 3T3 cells infected with the Oct4 and c-Myc retroviruses. Resistant NIH3T3 cell clones were screened using G418. Titers were measured using the Colony Forming Unit. Real infectious titers were 8.83×107cfu/mL and 5.50×10'cfu/mL respectively. These data revealed that very high titers infectious retroviruses generated from PT67 packaging cells.(3) Reprogramming of adult bovine skin fibroblasts into induced pluripotent stem cells with four factors.As previously reported, we splited the four factors combinations Oct4, Sox2, c-Myc, Klf4 to transfect the BDFs. To verify the characteristics of the bovine iPSCs, we determined the expression of ES cell-specific surface antigens and genetic markers by RT-PCR, qPCR, immunocytochemistry and western bloting. The biPSCs were strongly positive for AP, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Nanog, Oct4, Sox2, and TERT proteins. The biPSC colonies stained weakly for SSEA-1. Two CpG-enriched regions conserved in bovine were selected for analysis. The results demonstrated that Oct4 and Nanog promoter regions were highly unmethylated in bovine iPSC colonies. Bivine iPS cells showed exponential growth in culture, according to statistics,1.5×1011 and 2.4x1011 cells were acquired in 80 days. Karyotype analysis revealed that the bovine iPS cells had a normal 60 XX karyotpe.(4) Test for pluripotency of bovine induced pluripotent stem cellsbiPSCs effectively formed ball-shaped structures. We transferred these embryoid body-like structures to gelatin-coated plates and continued cultivation for another 10 days. These attached cells differentiated randomly into cells with various morphologies, such as resembling neuronal outgrowths, fibroblast-like cells and epithelial cells. Immunocytochemistry detected differentiated cells positive forβⅢ-tubulin, Nestin, GFAP, a-actin, Vimentin, Desmin, CK7, AFP and PDX-1, These data indicated that iPSCs can independently differentiate into lineages from all three primary embryonic germ layers in vitro.The bovine iPS cells were injected into dorsal of nonobese diabetes/severe-combined immunodeficient (NOD/SCID) mice. About 6 weeks after injection, we observed tumor formation, then tumors were collected and histological examined. The results showed that the teratomas contained various structures and tissues, including neural rosette, striated muscle, cartilage, vascular epithelium, and gut-like epithelium. These data revealed that biPSCs possess multilineage potential in vivo.(5) Effection of generation bovine iPS cells without proto-oncogeneWhen use of three specific transcription factors, Oct4, Sox2 and KLF4, iPS-like reprogramming cells can be acquired, but appeared to significantly reduce the efficiency of reprogramming(P<0.01). These cells can express the transcription factor and surface antigen, including OCT4, SOX2, NANOG, SSEA-3 and SSEA-4 by RT-PCR and immunocytochemistry, but the expression of TERT was not detected. In vitro, positive forβⅢ-tubulin, Vimentin, Desmin and CK7were detected in differentiated cells by immunocytochemistry, but the expression of AFP and Nestin absented by RT-PCR. Teratoma formation by these cells was observed striated muscle and vascular structures only. These data revealed that reprogramming cells by 3 factors were capable of differentiating into various cell types but it's not all. And use of two or one transcription factors, we can't acquire reprogramming cells in current condition.