Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-beta geo cassette.
We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre-mutant lox system, we successfully replaced the beta geo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp-deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the beta geo gene. Thus, using this pU-17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain-of-function mutation by replacing the beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.
Taniwaki
Takuya
T
Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan.
Haruna
Kyoko
K
Nakamura
Hiroshi
H
Sekimoto
Tomohisa
T
Oike
Yuichi
Y
Imaizumi
Takashi
T
Saito
Fumiyo
F
Muta
Mayumi
M
Soejima
Yumi
Y
Utoh
Ayako
A
Nakagata
Naomi
N
Araki
Masatake
M
Yamamura
Ken-ichi
K
Araki
Kimi
K
D000818:Animals; D001483:Base Sequence; D019295:Computational Biology; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006652:Histological Techniques; D051379:Mice; D008969:Molecular Sequence Data; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D013234:Stem Cells
D017931:DNA Primers; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D005819:Genetic Markers; D022821:RNA Splice Sites; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase