Backgrounds
Lentivirus vectors provide a delivery system that can both transduce nondividing cells and integrate transgenes into the genome of target cells which enable long-term tracing in vitro and in vivo. However, their transduction efficiency and stability of gene expression has not been evaluated in murine embryonic stem cells (ESC) or induced pleuripotent stem cells (iPS). Therefore, we evaluated lentiviral transduction into ESC or iPS and its gene expression.
Methods
Human immunodeficiency virus type 1-based lentivirus vectors containing the green fluorescent protein (GFP) gene were transduced into the cells with or without coating the culture dishes with retronectin. The effieciency of repeated transduction were compared with single transduction. The expression of GFP under the control of various promoter such as VEGFR2, eNOS, SM22 alpha, alpha myosin heavy chain were compared with that under a constitutively active promoter. Embryonic body (EB) from the transduced ESC and iPS was formed by hanging drop method. Spontaneous differentiation was induced by re-attaching the EB to fibronectin-coated dished. GFP expression was examined during differentiation.
Results
Following single transduction for 12h without retronectin and 2 days of culture, 27.3±6.7% of iPS were positive for GFP. Retronectin significantly increased the positivity of GFP (42.0±5.5%. n=4, p<0.001). Repeated transduction (3 times) elevated the positivity upto 95±3.4%. There was no difference of the transduction rate between iPS and ESC. There were relatively low expression of GFP under the control of VEGFR2, eNOS and SM22 alpha promoters whereas no expression under the control of alpha myosin heavy chain promoter. Seven days after spontaneous differentiation of the >90% GFP-positive iPS EB, GFP positivity was significantly reduced to 39.3±9.4%.
CONCLUSIONS: These data show that lentivirus with retronectin precoating and repreated transduction increases the efficiency of gene transfer in murine iPS or ESC. This method, however, may not be useful for the research in cardiovascular differentiation from iPS or ESC due to the significant gene silencing after differentiation.
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