Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy.
Recombinase-mediated unidirectional DNA inversion and transcriptional arrest is a promising strategy for high throughput conditional mutagenesis in the mouse. Banks of mouse embryonic stem cells with defined, transcriptionally silent insertions that can be activated by Cre recombinase would take advantage of existing transgenic Cre lines to rapidly produce hundreds of lineage specific and temporally controlled knockout mice for each gene, thereby introducing significant parallelism to functional gene annotation. However, the extent to which this strategy results in effective gene knockout has not been established. To test the feasibility of this strategy we targeted ErbB3, a member of the ErbB family of tyrosine kinase receptors, using this strategy. Insertion of a reversed "flipflox" vector consisting of a gene inactivation cassette (GI) and an internal ribosome entry site (IRES)-GFP reporter into intron 1 of ErbB3 was transcriptionally silent and did not affect ErbB3 expression. Crosses with ubiquitous and lineage specific Cre recombinase expressing lines permanently inverted the inserted GI cassette and blocked ErbB3 expression. Unidirectional DNA inversion by in vivo recombination is an effective strategy for targeted or ubiquitous gene knockout.
Qu
Shimian
S
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6838, USA. shimian.qu@vanderbilt.edu
Rinehart
Cammie
C
Wu
Hsiao-Huei
HH
Wang
Shizhen Emily
SE
Carter
Bruce
B
Xin
Hongbo
H
Kotlikoff
Michael
M
Arteaga
Carlos L
CL
D000818:Animals; D004247:DNA / Q000737:chemistry*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005260:Female; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D015151:Immunoblotting; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D049452:Green Fluorescent Proteins; D004247:DNA; D020893:Receptor, ErbB-3; C045073:Cre recombinase; D019426:Integrases