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RAPID COMMUNICATION |
a Department of Surgery, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom;
b School of Animal and Microbial Sciences, University of Reading, Reading, United Kingdom
Key Words. Human embryonic stem cells • Fluorescent reporter genes • Green fluorescent protein • Red fluorescent protein • Transfection • Small interfering RNA • Gain-of-function • Loss-of-function • Stem cell therapy • Regenerative medicine
Ludovic Vallier, Ph.D., Box 202, Level E9, Addenbrooke’s Hospital, Hills Road, Cambridge, CB2 2QQ, United Kingdom. Telephone: 44-1223-762025; Fax: 44-1223-410772; e-mail: lv225{at}cam.ac.uk
It is widely recognized that gain- and loss-of-function approaches are essential for understanding the functions of specific genes, and such approaches would be particularly valuable in studies involving human embryonic stem (hES) cells. We describe a simple and efficient approach using lipofection to transfect hES cells, which enabled us to generate hES cell lines expressing naturally fluorescent green or red proteins without affecting cell pluripotency. We used these cell lines to establish a means of diminishing gene function using small interfering (si)RNAs, which were effective at knocking down gene expression in hES cells. We then demonstrated that stable expression of siRNA could knock down the expression of endogenous genes. Application of these gain- and loss-of-function approaches should have widespread use, not only in revealing the developmental roles of specific human genes, but also for their utility in modulating differentiation.
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