Efficient target-selected mutagenesis in zebrafish.
One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.
Wienholds
Erno
E
Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
van Eeden
Freek
F
Kosters
Marit
M
Mudde
Josine
J
Plasterk
Ronald H A
RH
Cuppen
Edwin
E
D000818:Animals; D003433:Crosses, Genetic; D017931:DNA Primers / Q000138:chemical synthesis; D004334:Drug Administration Schedule; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005260:Female; D015723:Gene Library; D008297:Male; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D017354:Point Mutation; D015027:Zebrafish / Q000235:genetics*
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D010940:Plant Proteins; D005020:Ethyl Methanesulfonate