Cloning of bovine sox2 gene and construction of its retrovirus vector.
- Author:
Xiaoling XIN
1
;
Changrong LÜ
;
Dongmei CHEN
;
Zhongying DOU
Author Information
1. Shaanxi Branch of National Stem Cell Engineering & Technology Center, Laboratory of Molecular Biology for Agriculture, College of Animal Medicine, Northwest A & F University, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cattle;
Cloning, Molecular;
Genetic Vectors;
genetics;
Mice;
NIH 3T3 Cells;
Open Reading Frames;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
Retroviridae;
genetics;
metabolism;
SOXB1 Transcription Factors;
biosynthesis;
genetics;
Stem Cells;
metabolism;
Transfection
- From:
Chinese Journal of Biotechnology
2009;25(10):1464-1469
- CountryChina
- Language:Chinese
-
Abstract:
In order to construct the recombinant retrovirus vector of bovine sox2 gene and obtain infectious retroviral particles, we successfully amplified the ORF (open reading frame) of bovine sox2 gene from the primodial genital ridges of bovine embryo by RT-PCR. The cDNA of ORF was subcloned to pMD18-T vectors and verified that its sequence was highly homologous to the GenBank counterpart (GenBank Accession No. NM-001105463) by sequencing. The correct fragment was digested by EcoR I/Bgl II from recombinant pMD18-T vector and inserted into the same restriction sites f retroviral vector pMSCVneo. We got recombinant retrovirus vector pMSCV-sox2 which was transfected into PT67 by lipofectamine 2000 with pMIG (including green fluorescence protein) as a control. Flow cytometry analysis showed that its transfected efficiency was 68.3%. Subsequently, we established the stable cell strain by G418 selection which could produce virus. Its viral titer was up to 8.16x10(7) CFU/mL. This greatly facilitates the further study of bovine induced pluripotent stem cells induced from bovine somatic cells by defined factors.
