A High-Throughput Method for Identifying N-Ethyl-N-Nitrosourea (ENU)-Induced Point Mutations in Zebrafish

Publisher Summary

This chapter focuses on the high-throughput method for identifying n-ethyl-n-nitrosourea (ENU). Prior to screening for ENU-induced mutations in zebrafish, a library is generated consisting of cryopreserved sperm isolated from the F1 progeny of ENU-mutagenized males. Although it is possible to screen for mutations in fish that are kept alive, a cryopreserved library has several advantages. The zebrafish has become an important model system for vertebrate biology. Although forward genetic screens have uncovered the functions of many zebrafish genes, until recently no reliable, inexpensive, and high-throughput technology for targeted gene disruptions was developed. Zebrafish forward genetic screens have been—and continue to be—exceptionally productive. However, as the content of the zebrafish genome becomes available in the form of primary sequence information, it becomes increasingly evident that many essential genes have not been identified by this approach. Mutant phenotypes might be subtle or even undetectable in forward genetic screens because of the nature of the screen. For example, most genetic screens performed to date have focused on identifying phenotypes during the embryonic period while the embryo is still transparent and have therefore been easy to screen for morphological defects in the light microscope or following staining with tissue-specific markers.

Introduction

The zebrafish has become an important model system for vertebrate biology. Although forward genetic screens have uncovered the functions of many zebrafish genes, until recently no reliable, inexpensive, and high-throughput technology for targeted gene disruptions was developed. Here we outline an approach for identifying mutations in any gene of interest by TILLING (Targeting Induced Local Lesions in Genomes), a sensitive method for detecting single nucleotide polymorphisms (SNPs) in mutagenized genomes (Colbert 2001, McCallum 2000a, McCallum 2000b). This method uses the CEL1 assay to detect mutations in DNA isolated from N-ethyl-N-nitrosourea (ENU)-mutagenized F₁ individuals, which are heterozygous for randomly induced mutations (Fig. 1A). The CEL1 endonuclease specifically cleaves DNA 3′ to any single base pair mismatches present in heteroduplexes between wild-type and mutant DNA (Oleykowski et al., 1998). Genomic DNA isolated from mutagenized individuals is used as template for PCR amplification with gene-specific, fluorescently labeled (IRDye) primers. Because both wild-type and mutant alleles are amplified from heterozygous fish, heteroduplex PCR fragments are formed by denaturing and slowly reannealing the fragments. After CEL1 digestion, cleavage products are separated on a high-resolution polyacrylamide sequencing gel to reveal the presence and approximate location of induced mutations in the target sequence. This method allows the detection of rare ENU-induced mutations in the background of preexisting polymorphisms that are present even in inbred zebrafish strains.

To date, we have screened 25,303 kb from 5050 mutagenized genomes, using the approach described in this chapter, and have identified 48 new mutations (1 mutation per 527 kb screened). We anticipate, based on our data and data of others (Till 2003, Wienholds 2003) that about 5% of the mutations in coding DNA identified by TILLING will result in loss-of-function alleles. We therefore project that with DNA and frozen sperm from 10,000 individuals it should be possible to generate an allelic series that consists of on average, 20 mutations, including at least one loss-of-function and several hypomorphic alleles, provided the gene has an open reading frame that is ≥1 kb in size. We estimate that a library prepared as outlined later can be screened at least 50,000 times before the initial DNA resource is depleted, at which time more DNA can be isolated from reserve tissue.

The screening approach is outlined in Fig. 1B. Briefly, sperm collected from ENU-mutagenized F₁ adult males is cryopreserved in liquid nitrogen, using an efficient and rapid sperm cryopreservation protocol that archieves two sperm samples per F₁ male. Importantly, our sperm cryopreservation protocol allows the recovery of an average of 109 ± 84 (n = 46) viable F₂ progeny (or 28% ± 18% fertility) when one of the two samples is used for in vitro fertilization (see later). Genomic DNA is then purified from euthanized sperm donors, using a 96-well DNA isolation format. Next, mutants are identified with the CEL1 assay in a two-step screening process. For the primary screen, template DNA is pooled from 4 to 8 individuals, such that 384–768 mutagenized genomes are analyzed per 96-well plate of pooled DNA. PCR using IRDye-labeled primers and partial CEL1 digestion is performed in a 96-well format. Because the forward and reverse primers are labeled with unique fluorescent tags, it is possible to confirm mutations identified in one channel with the presence of the corresponding cleavage product in the second channel (Fig. 2). After identifying positive pools, mutant individuals are identified in a secondary CEL1 assay, and the nature of the mutation is determined by sequencing. Finally, fish lines carrying mutations that are likely to be deleterious are recovered by using the cognate cryopreserved sperm sample to fertilize eggs isolated from wild-type females (Fig. 1). The presence of the mutation in the F₂ generation is confirmed by PCR of tail-clipped DNA, using allele-specific primers or restriction fragment length polymorphism (RFLP) detection.

Our method differs in several ways from that of Wienholds et al. (2003):

• F₁ males are preserved as frozen sperm rather than as a living library. This eliminates the need for maintaining several thousand live fish during the screening process and provides a long-term (i.e., many-year) resource for mutation detection.

• Genomic DNA is normalized and pooled prior to PCR amplification rather than after PCR. This approach has allowed us to detect mutations efficiently in eightfold pooled samples rather than in fourfold pooled samples and thus decrease the expense of the mutation detection process (Draper, Moens, Till, Comai, and Henikoff, unpublished).

• A single-step PCR approach that uses gene-specific labeled primers is used rather than a nested PCR approach that uses universal labeled primers. Although this is more expensive in terms of primer cost, it requires less liquid handling capacity and therefore might be better suited to the smaller laboratory.

An example of the data produced by the CEL1 assay is presented in Fig. 2. In this example, a 443-bp fragment was screened for induced mutations in fourfold pools of template DNA. Following PCR amplification with IRDye-labeled primers, heteroduplexes were formed, digested with the CEL1 endonuclease, and separated on a LI-COR sequencing gel. The gel images generated for the IRD700 and IRD800 primer channels are shown in Fig. 2A and 2B, respectively. Bands present in multiple lanes identify preexisting polymorphisms in the target sequence, whereas unique bands present in one channel and having the corresponding cleavage product in the second channel (boxed in Fig. 2) indicate an induced mutation, one of which is visible on this gel.