Porcine Embryonic Oct4- EGFP Pluripotent Reporter Vector Construction

Oct4 is closely associated with embryonic development and differentiation,it belongs to POU transcription factor family. Oct4 is transcription factor expressed in mammalian embryonic cells which is also one of the most important cell pluripotency markers factor. Oct4 is specific expression in pluripotent cells as embryonic stem cells and primordial germ cells without expressing in differentiated somatic cells and play a key role in cell about maintaining pluripotency and self-renewal. Thus, Oct4 is often used as a marker to identify pluripotent cells. Controlling the expression of enhanced green fluorescent protein gene by the upstream regulatory region of Oct4 5,.Construct the Oct4-EGFP reporting system By observing the expression of green fluorescent protein to detect intracellular expression of Oct4, so we can study pluripotent cells in vivo conditions, Thus providing experimental evidence for early embryonic development and pig stem cell research.In this study, we activate the reconstructed embryos with CHX to impact on blastocyst rate of reconstructed embryos. The Oct4 promoter sequence to direct the recombinant vector pEGFP-N1 by the In-Fusion PCR Cloning technology, Oct4 promoter instead of the CMV promoter of the pEGFP-N1 plasmid to construct Oct4-EGFP reporter vector. The Oct4-EGFP reporter vector was transfected into Large White pigs embryonic by liposomal transfection to construction transgenic cell line with Oct4-EGFP reporter vector, the transgenic cell lines were injected into enucleated mature oocytes by somatic cell nuclear transfer technology, we observe EGFP expression in blastocysts to validation carrier. Results:(1) CHX was used and make a higher blastocyst rate than which without using(21.5%±2.1%VS11.5%±0.9%), Cleavage rate was not significantly different between them;(2)The In-Fusion PCR Cloning technology can efficiently construct Oct4-EGFP pluripotency reporter vector;(3)After liposomal transfection,,8 genetically modified cells of incorporates pluripotent report carrier with the Oct4-EGFP have been gained by screening and PCR;(4)Used 8 transgenic cell line with Oct4-EGFP reporter vectors as donor cells, we have got blastocysts by nuclear transfer method, and green fluorescence appeared in blastocysts, the Oct4-EGFP pluripotent reporter vector was transcribed and translated for green fluorescent protein in SCNT blastocyst pluripotent environment.Experimental results show that: Activation with CHX can significantly improve SCNT blastocyst rate of reconstructed embryos, Oct4 promoter-driven EGFP available at SCNT blastocysts express pluripotency environment and Oct4-EGFP reporter vector was efficacious.