Establishment of a porcine Oct-4 promoter-driven EGFP reporter system for monitoring pluripotency of porcine stem cells.
Porcine pluripotent cells with the capacity to generate germ line chimeras have not been developed yet. The transcription factor Oct-4 is an important marker of undifferentiating status and a central regulator of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system, such as that used in mice, will be a useful tool for monitoring the differentiating statuses of porcine cells both in vivo and in vitro. In the present study, we constructed a vector, pOGN2, in which enhanced green fluorescent protein (EGFP) was driven by the porcine Oct-4 promoter. In pigs containing this vector, EGFP was expected to be specifically expressed in pluripotent cells. We delivered the vectors into porcine fetal fibroblasts (PEFs) using liposomes. After transfected PEFs were selected with G418, we established eight cell lines containing the pOGN2 vector. When transgenic cells were used as donor nuclei to make somatic cell nuclear transfer (SCNT) embryos, SCNT embryos derived from four transgenic cell lines expressed green fluorescence. When PEFs with pOGN2 vectors were infected with retroviral vectors encoding the four transcription factors (Oct-4, Sox2, Klf4, and c-Myc), EGFP-expressing iPS cell colonies were observed at day 20. This work lays a foundation that can be used to generate a pig strain with an Oct4-EGFP reporter system, which would be greatly helpful in studying the differentiating and reprogramming mechanisms of pig embryos.
Institute of Comparative Medicine and Center of Experimental Animals, Southern Medical University, Guangzhou, People's Republic of China.
Lizhen
L
Huang
Nana
N
Fan
Jie
J
Cai
Dongshan
D
Yang
Bentian
B
Zhao
Zhen
Z
Ouyang
Weiwang
W
Gu
Liangxue
L
Lai
D000818:Animals; D054337:Cell Dedifferentiation; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012190:Retroviridae; D013552:Swine; D014157:Transcription Factors; D014161:Transduction, Genetic
C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins