Construction of recombinant proteins for reprogramming of endangered Luxi cattle fibroblast cells.
Generation of induced pluripotent stem cells from endangered cattle breeds makes a significant contribution to the establishment of embryonic stem cell line, conservation and utilization of genetic resources in endangered cattle. In our study, we are trying to construct recombinant proteins of transcription factors Oct-4, Nanog, Sox2 and Lin28 for reprogramming of endangered Luxi cattle fibroblast cells in induced pluripotent stem cells. To test the cell permeability and stability of the recombinant proteins, we designed and fused a pep-1 protein transduction domain to the C-terminus of enhanced green fluorescent protein, we treated Luxi cattle fibroblast cells with the purified proteins at various concentrations by adding them to the cell culture media for 2-48 h and assayed cell morphology and protein presence. We found that the purified EGFP-tagged recombinant proteins readily entered cells at a concentration of 0.12 mg/ml within 2 h and some of them could translocate into nucleus. In addition, we found that the transduced proteins appeared to be stable inside cells for up to 48 h. Then codons of Oct-4, Nanog, Sox2 and Lin28 were optimized for high level of expression in E. coli., they were synthesized using DNA oligo based, PCR gene assembling method, the reprogramming factors were designed with optimized transcription factors fused a pep-1 protein transduction domain to the C-terminus, for high level protein expression of the reprogramming factors, inducer concentration, and induction time were tested, the optimum inducer concentration for the expression of reprogramming factors Oct-4, Nanog, Sox2 and Lin28 were 0.01 mM, the optimum induction time were 10, 8, 2 and 12 h, respectively.
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
P F
PF
Hu
W J
WJ
Guan
X C
XC
Li
Y H
YH
Ma
D000818:Animals; D002417:Cattle; D054337:Cell Dedifferentiation; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003062:Codon; D056727:Endangered Species; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics; D010539:Permeability; D055550:Protein Stability; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics
D057846:Cell-Penetrating Peptides; D003062:Codon; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D055748:SOXB1 Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins