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.

Figure 1

Efficient target-selected mutagenesis in zebrafish. Forty male zebrafish were mutagenized with ENU and outcrossed with wild-type females to generate a library of 4608 mutagenized F₁ fish. Both males and females were finclipped and grouped in 384 pools of 12 fish per fish tank. DNA was isolated from the finclips and arrayed in twelve 384-well PCR plates. Amplicons of target genes were selected from local gene assemblies using GENOTRACE and primer picking software. Targets were amplified by PCR with gene-specific primers (1), followed by a nested PCR with internal gene-specific primers containing universal adaptor sequences (2), in combination with IR DYE-labeled universal M13 primers. Heteroduplexes were formed and samples were pooled fourfold. Pooled samples were incubated with CEL-I enzyme, and fragments were analyzed by denaturing polyacrylamide gel electrophoresis. Steps from PCR amplification to CEL-I incubation can be done in a completely unattended robotic setup. The four samples represented in a positive pool were reamplified from genomic DNA and subsequently resequenced. Fish carrying interesting mutations were recovered from pools of the F₁ library by finclipping and resequencing each of the 12 fish of a pool.

Figure 2

Representative example of a gel used to screen 384 fish for mutations in the dicer gene. Each of the 96 lanes of the polyacrylamide gel contains fourfold-pooled CEL-1-digested PCR products of ∼830 bp. The IR Dye 700 and IR Dye 800 channels are shown at left and right, respectively. Arrowheads indicate nine SNPs present in the founder fish. SNP no. 5 is only visible in one channel, and therefore, indicated by a gray arrowhead. Arrows indicate the three potential mutations on this gel (all rank 1).

Figure 3

Characterization of the ENU-induced mutations. (A) ENU mutation spectrum of this screen is similar to that of resequenced genes (Wienholds et al. 2002; E. Wienholds and R.H.A. Plasterk, unpubl.) and forward screens (Knapik 2000; http://zfin.org). Single nucleotide changes are indicated, and small deletions/insertions and chromosomal rearrangments are summarized under others. (B) Distribution of the mutations at the coding level.