TY - JOUR
T1 - Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype
AU - Kunath, Tilo
AU - Gish, Gerald
AU - Lickert, Heiko
AU - Jones, Nina
AU - Pawson, Tony
AU - Rossant, Janet
N1 - Funding Information:
Acknowledgments We thank Sue MacMaster and Lois Byers for tetraploid aggregations and uterine transfers and Andras Nagy for critical discussions. This research was supported by the Canadian Institutes of Health Research and a Terry Fox Programme Project grant from the National Cancer Institute of Canada.
PY - 2003/5/1
Y1 - 2003/5/1
N2 - Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.
AB - Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.
UR - http://www.scopus.com/inward/record.url?scp=0038708339&partnerID=8YFLogxK
U2 - 10.1038/nbt813
DO - 10.1038/nbt813
M3 - Article
C2 - 12679785
AN - SCOPUS:0038708339
SN - 1087-0156
VL - 21
SP - 559
EP - 561
JO - Nature Biotechnology
JF - Nature Biotechnology
IS - 5
ER -