Efficient Target-Selected Mutagenesis in Zebrafish

Abstract

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

Zebrafish: A model system for the study of eye genetics

“…Targeting induced local lesions in genomes (TILLING), a method originally developed for plant mutagenesis screens, has been used successfully to identify genetic lesions in specific genes of interest (McCallum et al, 2000;Wienholds et al, 2002Wienholds et al, , 2003Henikoff et al, 2004;Till et al, 2004). Although providing the opportunity to screen for mutations in virtually any gene, TILLING and similar approaches are very labor intensive, require largescale ENU mutagenesis combined with PCR-based assays to identify fish carrying lesions in the genes of interest, followed by the recovery of the alleles in subsequent generations.…”

How to make a teleost adenohypophysis: Molecular pathways of pituitary development in zebrafish

“…At the same time, genomic tools have enormously improved, facilitating the cloning of randomly induced mutations (Dahm and Geisler, 2006, Sivasubbu et al, 2007. In addition, methods of reverse genetics have been established, including gene knock-down via the injection of specific antisense morpholino oligos (MOs) (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003, target-selected ENU mutagenesis (also named TILLING; targeted induced local lesions in genomes) (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003, and, most recently, targeted introduction of small deletions via engineered zincfinger-nucleases (Nasevicius and Ekker, 2000, Doyon et al, 2008, Meng et al, 2008, Wienholds et al, 2003.…”

A protocol for TILLING and Ecotilling in plants and animals

“…TIL-LING is a reverse genetic strategy that combines traditional mutagenesis with high-throughput discovery of single-nucleotide changes 2,3 . The method is general and, following its original application to the model plant Arabidopsis thaliana, has been applied to a variety of plant and animal species including maize, lotus, barley, wheat, Drosophila and zebrafish [4][5][6][7][8][9] .…”