Effect of double gene transduction mediated by lentiviral vectors on characteristics of human embryonic stem cells / 中国病理生理杂志
Chinese Journal of Pathophysiology
;
(12): 2197-2203, 2009.
Article
in Chinese
| WPRIM
| ID: wpr-405481
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Chinese Journal of Pathophysiology
Year:
2009
Type:
Article
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