Enhancing and diminishing gene function in human embryonic stem cells.
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.
Vallier
Ludovic
L
Department of Surgery, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK. lv225@cam.ac.uk
Rugg-Gunn
Peter J
PJ
Bouhon
Isabelle A
IA
Andersson
Frida K
FK
Sadler
Amanda J
AJ
Pedersen
Roger A
RA
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods*; D015854:Up-Regulation / Q000235:genetics; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D000097573:Red Fluorescent Protein