A large-scale insertional mutagenesis screen in zebrafish.
It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.
Amsterdam
A
A
Biology Department and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Burgess
S
S
Golling
G
G
Chen
W
W
Sun
Z
Z
Townsend
K
K
Farrington
S
S
Haldi
M
M
Hopkins
N
N
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005799:Genes, Dominant; D005804:Genes, Lethal; D005808:Genes, Recessive; D005820:Genetic Testing; D008297:Male; D016254:Mutagenesis, Insertional; D009154:Mutation; D010641:Phenotype; D011533:Proviruses / Q000235:genetics; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics*
D017931:DNA Primers