| Transduction of eGFP Gene to Human Embryonic Stem Cells and Their Characterization. |
|
Yoon Young Kim, Seung Yup Ku, Yong Bin Park, Sun Kyung Oh, Shin Yong Moon, Young Min Choi |
1Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Korea.
2Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Korea.
3Central Research Institute, Sam Jin Pharm. Co. Ltd., Korea. |
|
|
| Abstract |
OBJECTIVE
Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. METHODS: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. RESULTS: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. CONCLUSION: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation haracteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies. |
| Key Words:
Human embryonic stem cells; Enhanced green fluorescent protein; Lentivirus |
|
PDF Links
Full text via DOI
Download Citation
