Effect of double gene transduction mediated by lentiviral vectors on characteristics of human embryonic stem cells
- VernacularTitle:慢病毒载体介导2次转基因对人胚胎干细胞特性的影响
- Author:
Liuhong CAI
;
Canquan ZHOU
;
Bin ZHANG
;
Xin TAO
;
Hui LIN
- Publication Type:Journal Article
- Keywords:
Human embryonic stem cells;
Lentiviral vector;
Gene transfection
- From:
Chinese Journal of Pathophysiology
2009;25(11):2197-2203
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To study the effect of double gene transduction mediated by lentiviral vectors on the characteristics of human embryonic stem cells (hESCs). METHODS: Using the backbone from inducibal dual and excisable transgene vector (iDuet101) , lentiviral vectors overexpressing cytotoxic T lymphocyte - associated molecule - 4 immuno-globulin (iDuet101 - CTLA4Ig) , indoleamine 2, 3 dioxygenase (iDuet101 - IDO) , and ubiquitin - C promoter - lueifer-ase - ires - puromycin ( ULIP) were constructed and packaged according to the standard recombinant techniques. Human embryonic stem cells (hESCs) were first transduced with iDuet101, iDuet101 - CTLA4Ig or iDuet101 - IDO, then, after the selection, were transduced again with ULIP. The expression and function of the exogenous genes were detected. Immu-nohistochemistry, RT - PCR and flow cytometry were applied for detection of embryoid bodies ( EB) formation in vitro and in vivo teratoma formation. RESULTS: Double - transduced hESCs showed typical shape of cell clones and positive staining of tumor rejection antigen -1 - 60 ( Tra -1 - 60 ) and octomer transcription factor - 4 ( OCT - 4 ). The formation of EB was observed, in which a - fetoprotein (AFP), paired box gene 6 ( Pax6) and Musashi 1 ( MSI1) were positively expressed. The cells formed teratomas, and the luciferase signals existed until 28 days after xeno - transplantation. CONCLUSION : Double transduction of non - transcriptional factors mediated by lentiviral vectors does not affect the cell growth rate and their differentiation ability.
