Efficient target-selected mutagenesis in Caenorhabditis elegans: toward a knockout for every gene

Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in genes of interest using high-throughput resequencing. A clonal library of 6144 EMS-mutagenized worms was established and screened, resulting in the identification of 1044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation of our results indicates that nonsense mutations for >90% of all C. elegans genes are present in the library. To identify all of these mutations, one only needs to inspect those positions that--given the known specificity of the mutagen--can result in the introduction of a stop codon. We define these positions as nonsense introducing mutations (NIMs). The genome-wide collection of possible NIMs can be calculated for any organism with a sequenced genome and reduces the screening complexity by 200- to 2000-fold, depending on the organism and mutagen. For EMS-mutagenized C. elegans, there are only approximately 500,000 NIMs. We show that a NIM genotyping approach employing high-density microarrays can, in principle, be used for the genome-wide identification of C. elegans knockouts.     

Insertional mutagenesis in zebrafish.

Insertional mutagenesis is a method for identifying genes essential for a given biological process by using the integration of DNA as the mutagen, thereby facilitating the cloning of the mutated gene. The use of retrovirus-mediated insertional mutagenesis in zebrafish has led to the mutation and rapid identification of hundreds of genes required for embryonic development and cell viability and growth, revealing the diversity of gene products required for the development of this vertebrate. Here, I will review the methodology of this approach and the results to date, as well as other potential ways to use insertional mutagenesis for genetic screens.     

A large-scale insertional mutagenesis screen in zebrafish

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.     

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.     

Perspectives in mutagenesis

I would first like to thank the Environmental Mutagen Society for honoring me with its 1980 award; I also would like to take this opportunity to acknowledge all the people who have worked with me and helped me since I started my work in mutagenesis in 1953. I feel especially indebted to my wife, Martha, whose support has been absolutely essential for my career. I would now like to explore some important problems in environmental mutagenesis. Obviously, I cannot touch upon new developments in all aspects of environmental mutagenesis - a field that has become increasingly broad in recent years. As in the past, there continue to be major advances in understanding mechanisms of mutagenesis, developing short-term tests, characterizing mutations, elucidating the relationship between mutagenesis and carcinogenesis, understanding the metabolism of mutagens, and many other areas. Many of these advances are reflected in the contents of the papers, symposia, and poster sessions of this meeting. In the years that I have been involved in mutation research and environmental mutagenesis, my interests and research activity have spanned a number of these areas; and I am pleased by the recent progress that I see taking place in them. In this talk, however, I would like to direct my comments toward a rather new dimension in environmental mutagenesis in which I have become increasingly interested: The monitoring of mutations in the human population. In the monitoring of the human population there are two choices: We can monitor the progeny of an exposed population or we can consider exposed persons as populations of cells and measure the mutant frequency in individual cells in these persons. Technical developments are advancing rapidly in both of these approaches. The pioneering work of James Neel in measuring germinal effects of mutagens in the human population is well known [Neel, 1970]. Methods for measuring mutation frequencies in single cells in vivo also are beginning to be available. For instance, Strauss and Albertini [1979] are developing a system to detect HGPRT mutants in human lymphocytes. Of course, there are still difficult problems to be resolved in this system; and we do not know with certainty.     

Complement system in zebrafish

Zebrafish is recently emerging as a model species for the study of immunology and human diseases. Complement system is the humoral backbone of the innate immune defense, and our knowledge as such in zebrafish has dramatically increased in the recent years. This review summarizes the current research progress of zebrafish complement system. The global searching for complement components in genome database, together with published data, has unveiled the existence of all the orthologues of mammalian complement components identified thus far, including the complement regulatory proteins and complement receptors, in zebrafish. Interestingly, zebrafish complement components also display some distinctive features, such as prominent levels of extrahepatic expression and isotypic diversity of the complement components. Future studies should focus on the following issues that would be of special importance for understanding the physiological role of complement components in zebrafish: conclusive identification of complement genes, especially those with isotypic diversity; analysis and elucidation of function and mechanism of complement components; modulation of innate and adaptive immune response by complement system; and unconventional roles of complement-triggered pathways.     

Somatic mutagenesis in autoimmunity

Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemic lupus erythematosus (SLE). SLE is associated with high titers of serum autoantibodies of the IgG class that are predominantly directed against nuclear antigens, with pathological manifestations that are considered by many to be characteristic of an immune-complex mediated disease. In this review, we focus on the known and potential roles of somatic mutagenesis in SLE. We will argue that anti-nuclear antibodies (ANA) arise predominantly from nonautoreactive B cells that are transformed into autoreactive cells by the process of somatic hypermutation (SHM), which is normally associated with affinity maturation during the germinal center reaction. We will also discuss the role of SHM in creating antigenic peptides in the V region of the B cell receptor (BCR) and its potential to open an avenue of unregulated T cell help to autoreactive B cells. Finally, we will end this review with new experimental evidence suggesting that spontaneous somatic mutagenesis of genes that regulate B cell survival and activation is a rate-limiting causative factor in the development of ANA.     

Transposon mutagenesis in Halomonas eurihalina

We have established a transposon mutagenesis procedure for the moderate halophile Halomonas eurihalina, a bacteria that produces an exopolysaccharide (EPS) of considerable biotechnological interest. We used suicide plasmids pUT and pSUP102 to introduce the transposons mini-Tn5 and Tn1732 into H. eurihalina via Escherichia coli mediated conjugation. Southern hybridization analysis demonstrated that insertions of the transposon mini-Tn5 into H. eurihalina occurred randomly at single sites in the chromosome, whereas Tn1732 insertion also took place at random, but simultaneously, at several sites. Phenotypic analysis revealed that different mutants were generated by using mini-Tn5. The isolation of exopolysaccharide-defective strains is the first stage towards carrying out genetic studies on EPS production by this microorganism.     

Studies in comparative chemical mutagenesis

This paper presents a review of various collaborative studies in comparative mutagenesis. The following studies are briefly described: (1) the chemical mutagenesis programme of the European Community, (2) Drosophila studies with various alkylating agents of different s (Swain-Scott) factors, (3) the evaluation by the International Commission for Protection against Environmental Mutagens and Carcinogens (ICPEMC) Committee 1, (4) the Environmental Protection Agency's Gene-Tox Programme, (5) the first and second United Kingdom Environmental Mutagen Society (UKEMS) collaborative studies, and (6) the International Programme on Chemical Safety (IPCS) collaborative study on in vitro tests. The need for chemical dosimetry is emphasized. One of the main conclusions is that, of the mammalian point mutation assays, the L5178Y (TFTR, trifluorothymidine resistant) system showed greatest detection capability in the second UKEMS study. The consensus conclusion of the IPCS in vitro study was that chromosomal aberrations are considered to be the optimal assay for complementing the Salmonella assay and offer the additional advantage that aneuploidy, polyploidy, and sister chromatid exchanges can also be easily assessed.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae

Summary Four types of mit ^– mutations induced with manganese are found in the following relative proportions: oxi3 ^– > cob-box ^– > oxi2 ^– oxi1 ^–1. The frequences of loss of their respective mit ⁺ alleles in manganese-induced rho ^–] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.     

Somite development in zebrafish.

A full understanding of somite development requires knowledge of the molecular genetic pathways for cell determination as well as the cellular behaviors that underlie segmentation, somite epithelialization, and somite patterning. The zebrafish has long been recognized as an ideal organism for cellular and histological studies of somite patterning. In recent years, genetics has proven to be a very powerful complementary approach to these embryological studies, as genetic screens for zebrafish mutants defective in somitogenesis have identified over 50 genes that are necessary for normal somite development. Zebrafish is thus an ideal system in which to analyze the role of specific gene products in regulating the cell behaviors that underlie somite development. We review what is currently known about zebrafish somite development and compare it where appropriate to somite development in chick and mouse. We discuss the processes of segmentation and somite epithelialization, and then review the patterning of cell types within the somite. We show directly, for the first time, that muscle cell and sclerotome migrations occur at the same time. We end with a look at the many questions about somitogenesis that are still unanswered.     

Genetic variation in the zebrafish

Although zebrafish was introduced as a laboratory model organism several decades ago and now serves as a primary model for developmental biology, there is only limited data on its genetic variation. An establishment of a dense polymorphism map becomes a requirement for effective linkage analysis and cloning approaches in zebrafish. By comparing ESTs to whole-genome shotgun data, we predicted >50,000 high-quality candidate SNPs covering the zebrafish genome with average resolution of 41 kbp. We experimentally validated approximately 65% of a randomly sampled subset by genotyping 16 samples from seven commonly used zebrafish strains. The analysis reveals very high nucleotide diversity between zebrafish isolates. Even with the limited number of samples that we genotyped, zebrafish isolates revealed considerable interstrain variation, ranging from 7% (inbred) to 37% (wild-derived) of polymorphic sites being heterozygous. The increased proportion of polymorphic over monomorphic sites results in five times more frequent observation of a three allelic variant compared with human or mouse. Phylogenetic analysis shows that comparisons between even the least divergent strains used in our analysis may provide one informative marker approximately every 500 nucleotides. Furthermore, the number of haplotypes per locus is relatively large, reflecting independent establishment of the different lines from wild isolates. Finally, our results suggest the presence of prominent C-to-U and A-to-I RNA editing events in zebrafish. Overall, the levels and organization of genetic variation between and within commonly used zebrafish strains are markedly different from other laboratory model organisms, which may affect experimental design and interpretation.     

基于PCR的体外诱变技术

定点突变技术是蛋白质工程中应用的重要技术之一,可以有目的改变DNA序列中的碱基。它不仅可以阐明基因表达的调控机理,还可用研究蛋白质结构与功能间的关系,改造天然蛋白质,使之更符合应用需求。自从PCR技术发明以来,便被用于进行定点突变。综述了各种基于PCR技术的定点突变方法,并与其他定点突变方法进行对比。     

定点突变技术的研究进展

定点突变技术是蛋白质工程中采用的重要技术之一,可以有目的地改变DNA序列中的碱基;它不仅可以用来阐明基因的调控机理,还可以用来研究蛋白质结构与功能之间的关系。本文综述了3种常用的定点突变方法的原理及应用并对三者的优缺点和适用范围进行了比较。     

BMP regulation of myogenesis in zebrafish

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi‐nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation. Developmental Dynamics 239:806–817, 2010. © 2010 Wiley‐Liss, Inc.