Construction of Nanog lentiviral expression vector and its application in differentiation regulation of embryonic stem cells
10.3969/j.issn.2095-4344.2016.19.004
- VernacularTitle:Nanog慢病毒载体构建及在胚胎干细胞分化调控中的应用
- Author:
Fei CHEN
;
Li WAN
;
Yan LI
;
Xin LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2016;20(19):2763-2769
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:There is a lack of safe and effective modular lentivectors in differentiation regulation of embryonic stem cel s. OBJECTIVE:To construct a modular lentivector, Puro-Nanog-hrGFP, with Nanog promoter-control ed humanized renil a reniformis green fluorescent protein (hrGFP) and puromycin and to observe the expression of Nanog during the differentiation of Nanog-hrGFP-transfected mouse embryonic stem cel lines. METHODS:After PCR amplification, Nanog, hrGFP and entry vector were recombined into the pDest-puro vectors to generate the lentiviral expression vector, Puro-Nanog-hrGFP, by the LR reaction. Through lentivirus production, transduction and puromycin screening, the transduced cel lines with Nanog-hrGFP gene were generated and identified. Expression of Nanog during the differentiation of transgenic mouse cel lines was detected. RESULTS AND CONCLUSION:The lentiviral expression vectors Puro-Nanog-hrGFP were constructed successful y by Gateway technology, and then the transducted cel lines were obtained by lentiviral infection. The expression of Nanog was gradual y decreased during the process of transgenic cel lines differentiation, which provides a new tool for further investigation on regulation of stem cel differentiation.
