A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.
We report here development of a novel gene trap method in zebrafish using the Tol2 transposon system. First, we established a highly efficient transgenesis method in which a plasmid DNA containing the Tol2 transposon vector and the transposase mRNA synthesized in vitro were coinjected into one-cell stage embryos. The transposon vector inserted in the genome could be transmitted to the F1 progeny at high frequencies, and regulated gene expression by a specific promoter could be recapitulated in transgenic fish. Then we constructed a transposon-based gene trap vector containing a splice acceptor and the GFP gene, performed a pilot screen for gene trapping, and obtained fish expressing GFP in temporally and spatially restricted patterns. We confirmed the endogenous transcripts were indeed trapped by the insertions, and the insertion could interfere with expression of the trapped gene. We propose our gene trap approach should facilitate studies of vertebrate development and organogenesis.
Kawakami
Koichi
K
Division of Molecular and Developmental Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan. kokawaka@lab.nig.ac.jp
Takeda
Hisashi
H
Kawakami
Noriko
N
Kobayashi
Makoto
M
Matsuda
Naoto
N
Mishina
Masayoshi
M
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000494:pharmacology; D014158:Transcription, Genetic / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D019895:Transposases / Q000235:genetics; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; D008164:Luminescent Proteins; D012333:RNA, Messenger; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins; D019895:Transposases