Cloning and expression of human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells

• Published in: Xi bao yu fen zi mian yi xue za zhi Vol. 32; no. 3; p. 308
• Main Authors: Ruan, Bibo, Fu, Wuyu, Rui, Wen, Chen, Haixuan, Zhou, Pengjun, Liao, Shuangye, Wu, Xiaoqing, Wang, Yifei, Chen, Hongyuan
• Format: Journal Article
• Language: Chinese
• Published: China 01-03-2016
• Subjects: Blotting, Western; Cloning, Molecular - methods; DNA, Complementary - genetics; Escherichia coli - genetics; Gene Expression; Genetic Vectors - genetics; HEK293 Cells; HeLa Cells; Humans; Octamer Transcription Factor-3 - genetics; Octamer Transcription Factor-3 - metabolism; Plasmids - genetics; Recombinant Proteins - metabolism
• ISSN: 1007-8738

To clone and express human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells.
Primers were designed based on the coding sequence for the human Oct4 cDNA. The fragment containing Oct4 gene, amplified by reverse transcription PCR (RT-PCR) from human HeLa cell cDNA as the template, was subcloned into pET-22b(+) and pcDNA3.1(+) vectors to construct the gene recombinant prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)-his-Oct4, respectively. Thereafter, the double enzymes digestion and the target gene sequencing were performed for identification. The prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)- his-Oct4 from the positive clones were respectively transformed into E.coli BL21 (DE3) and the embryo HEK293T cells for protein expression.
DNA fragments (1100 bp) containing the coding region of Oct4 gene were obtained from RT-PCR. The recombinant expression plasmids were confirmed by enzymes digestion identif