A whole lotta jumpin' goin' on: new transposon tools for vertebrate functional genomics

Genome sequences of vertebrate model organisms are becoming available, fuelling the next challenging phase of research: annotating all of the genes with functional information. The functional annotation of all of the approximately 35 000 genes in mammals will undoubtedly require complementing the technologies of forward and reverse genetics in mutagenesis screens. Two recent articles report on the construction of retrotransposable elements that efficiently 'jump' in mammalian cells. These new elements hold great promise as useful vectors for insertional mutagenesis screens in the mouse.     

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.     

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.     

Transposition of the Drosophila hydei Minos transposon in the mouse germ line

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.     

N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

Manipulating the <Emphasis Type="Italic">Xenopus</Emphasis>genome with transposable elements

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.     

DNA转座子在小鼠基因功能研究中的应用

近年来,转座子介导的插入突变在哺乳动物的分子遗传学研究中得到了广泛的应用。转座子作为一种简便高效的遗传学操作工具,在构建转基因动物模型、基因治疗、细胞水平和动物整体水平的基因功能研究等方面发挥了重要的作用。文章重点介绍DNA转座子的结构、功能及其应用于小鼠分子遗传学领域的最新研究进展。     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

插入突变在水稻功能基因组学中的研究进展

构建饱和的基因突变体库是最直接、有效的分析鉴定基因功能的方法.根据插入突变源不同可分为T-DNA插入突变、转座子插入突变等.主要介绍这两种方法的原理及其在水稻功能基因组学研究中的应用和进展,并分析和讨论了插入突变在水稻功能基因组学研究中存在的困难和发展趋势.     

The expanding role of mouse genetics for understanding human biology and disease

It has taken about 100 years since the mouse first captured our imagination as an intriguing animal for it to become the premier genetic model organism. An expanding repertoire of genetic technology, together with sequencing of the genome and biological conservation, place the mouse at the foremost position as a model to decipher mechanisms underlying biological and disease processes. The combined approaches of embryonic stem cell-based technologies, chemical and insertional mutagenesis have enabled the systematic interrogation of the mouse genome with the aim of creating, for the first time, a library of mutants in which every gene is disrupted. The hope is that phenotyping the mutants will reveal novel and interesting phenotypes that correlate with genes, to define the first functional map of a mammalian genome. This new milestone will have a great impact on our understanding of mammalian biology, and could significantly change the future of medical diagnosis and therapeutic development, where databases can be queried in silico for potential drug targets or underlying genetic causes of illnesses. Emerging innovative genetic strategies, such as somatic genetics, modifier screens and humanized mice, in combination with whole-genome mutagenesis will dramatically broaden the utility of the mouse. More significantly, allowing genome-wide genetic interrogations in the laboratory, will liberate the creativity of individual investigators and transform the mouse as a model for making original discoveries and establishing novel paradigms for understanding human biology and disease.     

Transposable elements in fish functional genomics: technical challenges and perspectives

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.

     

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

Checks and balancers: balancer chromosomes to facilitate genome annotation

Phenotype-driven mutagenesis screens are used to discover gene function in model organisms. Mutations that are induced by chemical mutagens can occur anywhere in the genome. However, the use of a balancer chromosome (where a phenotypically marked segment of a chromosome is inverted) in a mutagenesis screen enables mutations to be mapped in a defined region of the genome and maintained stably in a heterozygous state. Mouse balancer chromosomes can be engineered using Cre-loxP technology in selected regions of the genome. Balancer mutagenesis screens will provide a systematic functional analysis of the genes on mouse chromosomes, and consequently, will facilitate a functional annotation of the mammalian genome sequence.     

Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice

Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.     

Novel lethal mouse mutants produced in balancer chromosome screens

Mutagenesis screens are a valuable method to identify genes that are required for normal development. Previous mouse mutagenesis screens for lethal mutations were targeted at specific time points or for developmental processes. Here we present the results of lethal mutant isolation from two mutagenesis screens that use balancer chromosomes. One screen was localized to mouse chromosome 4, between the STS markers D4Mit281 and D4Mit51. The second screen covered the region between Trp53 and Wnt3 on mouse chromosome 11. These screens identified all lethal mutations in the balancer regions, without bias towards any phenotype or stage of death. We have isolated 19 lethal lines on mouse chromosome 4, and 59 lethal lines on chromosome 11, many of which are distinct from previous mutants that map to these regions of the genome. We have characterized the mutant lines to determine the time of death, and performed a pair-wise complementation cross to determine if the mutations are allelic. Our data suggest that the majority of mouse lethal mutations die during mid-gestation, after uterine implantation, with a variety of defects in gastrulation, heart, neural tube, vascular, or placental development. This initial group of mutants provides a functional annotation of mouse chromosomes 4 and 11, and indicates that many novel developmental phenotypes can be quickly isolated in defined genomic intervals through balancer chromosome mutagenesis screens.     

Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project

The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes.     

A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice.

Gene targeting in mouse embryonic stem (ES) cells can be used to generate single gene mutations or defined multi-megabase chromosomal rearrangements when applied with the Cre- loxP recombination system. While single knockouts are essential for uncovering functions of cloned genes, chromosomal rearrangements are great genetic tools for mapping, mutagenesis screens and functional genomics. The conventional approach to generate mice with targeted alterations of the genome requires extensive molecular cloning to build targeting vectors and DNA-based genotyping for stock maintenance. Here we describe the design and construction of a two-library system to facilitate high throughput gene targeting and chromo-somal engineering. The unique feature of these libraries is that once a clone is isolated, it is essentially ready to be used for insertional targeting in ES cells. The two libraries each bear a complementary set of genetic markers tailored so that the vector can be used for Cre- loxP -based chromosome engineering as well as single knockouts. By incorporating mouse coat color markers into the vectors, we illustrate a widely applicable method for stock maintenance of ES cell-derived mice with single gene knockouts or more extensive chromosomal rearrangements.     

Developing genetic reagents to facilitate recovery, analysis, and maintenance of mouse mutations

Because the mouse has become the pre-eminent model system for functional genomics and analysis of complex-systems/pathways in mammals, there has been an escalation of interest in the generation and analysis of mouse mutations to use as tools in these analyses. I argue here for a parallel investment in continuing the development of appropriately marked chromosomal rearrangements to use as genetic reagents in mutation recovery, analysis, and maintenance crosses. Specifically, visibly marked interstitial chromosomal deletions can be valuable for regional mutagenesis screens for recessives based on hemizygosity, and they can also be used to simplify genetic fine-mapping as a prelude to gene identification based on positional cloning/candidacy strategies. Dominantly marked chromosomal inversions that also manifest some kind of recessive phenotype can be exploited in more extensive regional mutagenesis screens based on homozygosity, and are invaluable for simplified, low-cost and error-reduced mutant-stock maintenance. Also discussed are several issues concerning genetic background, particularly from the point of view of genetic-reagent resource development. Received: 16 December 1999 / Accepted: 16 December 1999