Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank.
To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.
Zwaal
R R
RR
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Broeks
A
A
van Meurs
J
J
Groenen
J T
JT
Plasterk
R H
RH
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015723:Gene Library; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009348:Nematoda / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction
D004251:DNA Transposable Elements; D009838:Oligodeoxyribonucleotides; D004247:DNA