Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice.
Conditional gene targeting strategies are progressively used to study gene function tissue-specifically and/or at a defined time period. Instrumental to all of these strategies is the generation of targeting vectors, and any methodology that would streamline the procedure would be highly beneficial. We describe a comprehensive transposition-based strategy to produce gene-targeting vectors for the generation of mouse conditional alleles. The system employs a universal cloning vector and two custom-designed mini-Mu transposons. It produces targeting constructions directly from BAC clones, and the alleles generated are modifiable by Cre and Flp recombinases. We demonstrate the applicability of the methodology by modifying two mouse genes, Chd22 and Drapc1. This straightforward strategy should be readily suitable for high-throughput targeting vector production.
Turakainen
Hilkka
H
Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland.
Saarimäki-Vire
Jonna
J
Sinjushina
Natalia
N
Partanen
Juha
J
Savilahti
Harri
H
D000483:Alleles; D000818:Animals; D001287:Attachment Sites, Microbiological; D015820:Cadherins / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D019426:Integrases / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
C498341:Apcdd1 protein, mouse; D015820:Cadherins; C504494:Cdh22 protein, mouse; D004251:DNA Transposable Elements; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases