Engineering mutations: Deconstructing the ... - Wiley Online Library

DEVELOPMENTAL DYNAMICS 235:2424 –2436, 2006

SPECIAL ISSUE REVIEWS–A PEER REVIEWED FORUM

Engineering Mutations: Deconstructing the Mouse Gene by Gene Christopher S. Raymond and Philippe Soriano*

Over the past years new vectors and methodologies have been developed to carry out large-scale genomewide insertional mutagenesis screens in the mouse. Gene trapping, the most commonly used technique, is based on the insertion of a retroviral- or plasmid-based vector into a gene, resulting in a loss-of-function mutation, while simultaneously reporting its expression pattern and providing a molecular tag to facilitate cloning. The discovery of vertebrate DNA transposons in the mouse and recent improvements has also led to their increased use in insertional mutagenesis screens. Several public resources have been set-up recently by the academic community to distribute information and materials generated from these largescale screens. These new resources should accelerate the study and understanding of biological and developmental processes. Developmental Dynamics 235:2424 –2436, 2006. © 2006 Wiley-Liss, Inc. Key words: insertional mutagenesis; gene trap; transposon; genetic screen; mouse Accepted 17 April 2006

INTRODUCTION With the completion of the sequencing of the mouse and human genomes, we now face the major challenge of understanding the function of each of the estimated 25,000 genes. Due to its high overall similarity to humans and facility for genetic manipulation, the laboratory mouse (Mus musculus) is the best mammalian model organism system in which to define gene function that underlies human disease. Several large-scale mutagenesis programs, similar to those that have been carried out with great success in Drosophila and Caenorhabditis elegans, are currently under way in the mouse to identify and assign gene function. The focus of this review is to give an overview of insertional mutagenesis methodologies in the mouse. Various screening strategies will be discussed, highlighting new genetic tools and

public resources that are available to help accelerate the task of assigning function to mammalian genes.

STRATEGIES FOR MUTAGENESIS Three common methods have been used to introduce mutations into the mouse germline: radiological (Green and Roderick, 1966; Rinchik, 1991), chemical (Russell et al., 1979, 1989; Munroe et al., 2000), and insertional (Schnieke et al., 1983; Wagner et al., 1983; Jenkins and Copeland, 1985; Robertson et al., 1986; Doetschman et al., 1987; Thomas and Capecchi, 1987; Gossler et al., 1989; Spence et al., 1989) mutagenesis. Radiological methods, usually involving the exposure of mice to X-rays, lead to chromosomal rearrangements ranging from simple small deletions to more com-

plex rearrangements, such as large inversions or translocations (Green and Roderick, 1966). A benefit of large DNA rearrangements is that they can be used for mapping and identifying regions of interest in the mouse genome. However, these large rearrangements can also be a drawback. Because multiple genes are usually affected in large chromosomal rearrangements, it is difficult to relate the mutant mouse phenotype to the function of a single gene. Therefore, this type of approach does not lend itself well to high-throughput mutagenesis. Chemical mutagenesis provides an alternative means of introducing mutations into the mouse genome. Nethyl-N-nitrosourea (ENU) is the most commonly used chemical mutagenic agent in the mouse. When introduced intraperitoneally into the mouse, it can lead to thousands of mu-

Program in Developmental Biology, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington *Correspondence to: Philippe Soriano, Program in Developmental Biology, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109. E-mail: [email protected] DOI 10.1002/dvdy.20845 Published online 24 May 2006 in Wiley InterScience (www.interscience.wiley.com).

© 2006 Wiley-Liss, Inc.

MUTAGENESIS IN THE MOUSE 2425

Fig. 1. Schematic diagram of promoter and polyA gene trap vectors. Ai: Intron trap vector consisting of an SA sequence, selection/expression cassette, and polyA signal. Insertion into the intron of a transcriptionally active gene generates a fusion transcript that confers drug resistance and can also report the expression pattern of the endogenous trapped gene. Aii: Exon trap vector with selection/expression cassette and polyA signal but lacking an SA sequence inserts in-frame into an exon (or single exon-containing gene, as shown here), resulting in a fusion transcript that allows for drug selection and expression analysis. B: PolyA trap vector containing an SD sequence inserts into an intron of transcriptionally active gene capturing the endogenous polyA sequence resulting in a fusion transcript that confers drug resistance.

tations being transmitted through the germline (Russell et al., 1979). Most of these mutations are single base pair substitutions and occasionally small deletions, therefore, most likely affecting only a single gene (Popp et al., 1983). ENU mutagenesis does not always lead to a null mutation but may be used to create hypomorphs and allelic series (Chen et al., 2000; Vivian et al., 2002). This approach easily can generate thousands of random mutations across the mouse genome and, thus, is amenable for use in high throughput screening (for examples, see Hrabe de Angelis et al., 2000; Nolan et al., 2000; Kile et al., 2003). However, ENU-generated mutations can be difficult to map and laborious to identify, as there is no easy molecular tag. Another drawback of ENU mutagenesis is that large mouse colonies

must be maintained and extensive breeding performed to examine recessive phenotypes. Nonetheless, ENU mutagenesis has proven to be a popular and powerful approach that will be discussed in more depth elsewhere in this journal (Caspary and Anderson, 2006). Last, insertional mutagenesis provides an attractive approach for performing forward genetic screens in the mouse. Two advantages over the other methods mentioned above are the ability to generate null mutations and the ease with which a mutated gene can be identified. Insertional mutagenesis can be either achieved by gene targeting or gene trapping. Gene targeting by homologous recombination in mouse embryonic stem (ES) cells is a proven approach to elucidate the function of known genes

(Doetschman et al., 1987; Thomas and Capecchi, 1987). Approximately 4,000 mutations have been engineered in the mouse genome since this technique was developed in the late 1980s. However, this approach does not lend itself to forward genetic screens in the mouse, because it requires previous knowledge of the gene and its structure. In addition, large-scale gene targeting projects are complex due to the time-consuming and labor-intensive process of generating targeting vectors for each individual gene, although recent advances in recombineering have helped facilitate this process (for reviews, see Copeland et al., 2001, and http://recombineering.ncifcrf.gov/). Gene traps are another tool for producing tagged random mutations in the mouse genome, but unlike gene targeting, they require no previous knowledge of the gene structure (Friedrich and Soriano, 1991; von Melchner et al., 1992; Wurst et al., 1995; Hicks et al., 1997; Mitchell et al., 2001; Hansen et al., 2003; Zambrowicz et al., 2003). Although this method is not generally designed to disrupt a specific target gene, it can be used to generate a library of mutated cell lines. Gene traps were originally developed as an efficient technique to generate mutations in ES cells (Gossler et al., 1989; Friedrich and Soriano, 1991; von Melchner et al., 1992). Gene traps are retroviral-, plasmid-, or transposon-based vectors that disrupt the transcription of a gene upon insertion (for review see Stanford et al., 2001). There are many different types of gene trap vectors currently in use today; however, the two most basic and common types of gene traps are termed promoter traps and polyA traps. The most commonly used promoter trap vectors are intron traps that typically contain a splice acceptor sequence followed by a reporter gene, usually a promoterless ATG-containing ␤geo selection cassette (a fusion of neomycin phosphotransferase and ␤-galactosidase) and a polyadenylation signal (polyA; Fig. 1Ai; Gossler et al., 1989; Friedrich and Soriano, 1991). Upon integration within an intron of a gene, the ␤geo reporter gene is expressed and serves as a means for positive drug selection in vitro. Because the reporter cassette is also under the control of the endogenous re-

2426 RAYMOND AND SORIANO

porter, it can often reveal the expression pattern of the endogenous gene (Skarnes et al., 1992). If the gene trap inserts near the 5⬘ end of a gene, it is likely to result in a null or at least a severe hypomorphic allele. An important consideration is the choice of the splice acceptor sequence, as skipping of the gene trap artificial exon might lead to a hypomorphic mutation. Alternative splicing may be circumvented by limiting the length of the splice acceptor sequence, thus preventing the inclusion of cryptic splice sites. The fusion transcript generated by the trapped gene allows the gene identification by 5⬘-rapid amplification of cDNA ends (RACE; Frohman, 1993). Alternatively, the genomic DNA region flanking the insertion site can be cloned using an adaptor-mediated polymerase chain reaction (PCR) cloning method (Usman et al., 2000) or inverse PCR. The methodology for identification of gene trap insertions by RACE or cloning of genomic flanking sequence is covered in more detail in other method-based gene trap reviews (Friedrich and Soriano, 1993; Chen and Soriano, 2003). A second type of promoter trap, referred to as an exon trap, does not include a splice acceptor and is designed to disrupt gene activity when inserted in an exon (Fig. 1Aii; von Melchner et al., 1992; Hicks et al., 1997). This strategy is advantageous for trapping of genes containing a single exon; however, these vectors are inherently less efficient than intron traps for large-scale mutagenesis due to their smaller target sites and requirement for inserting in-frame to a gene. One of the shortcomings of these promoter trap vectors is that the gene must be expressed in the target cell. This caveat limits the types of genes that can be inactivated by this method. Trapping of genes that are not expressed in target cells requires the use of a third type of gene trap vector, known as a PolyA trap. PolyA traps feature a selectable reporter that is driven by a strong ubiquitous promoter (Fig. 1B; Niwa et al., 1993; Yoshida et al., 1995; Zambrowicz et al., 1998). These constructs replace the polyA signal with a splice donor sequence following the reporter gene. A polyA trap vector must insert into the intron of a gene in the correct orienta-

tion to splice with the downstream exons and acquire the endogenous polyA of the trapped gene. The mutagenicity of polyA trap vectors is debatable, however, as these vectors often have a bias to insert further toward the 3⬘end of genes, allowing for the generation of truncated proteins. A recent study suggests that skewing of insertions toward the 3⬘-end of a gene is due to nonsense-mediated mRNA decay (NMD) of the polyA trap fusion transcripts (Shigeoka et al., 2005). This group developed an improved polyA trap vector, UPATrap, that includes an internal ribosome entry site (IRES) sequence placed downstream of a termination codon-containing selectable marker. The authors found that inclusion of this IRES sequence suppresses the NMD of the selectable marker message and relieves the bias toward selecting for 3⬘-insertions, while still allowing for the trapping of genes that are not expressed in the target cells. The delivery of gene traps into cells can be achieved by means of retroviral- or plasmid-based vectors, with each modality having their advantages and disadvantages. Retroviruses display a preference toward integration at the 5⬘-end of the gene, often in the first intron (Wu et al., 2003; Chen et al., 2004a; De-Zolt et al., 2006). This finding may be an advantage for generating null alleles, as there will be little or no protein sequence anchored upstream of the ␤geo reporter. This insertion site preference may be due to an open chromatin configuration of the DNA in this region that facilitates retroviral integration (Wu et al., 2003). The multiplicity of infection with a retroviral gene trap also can be tightly controlled to deliver a single gene trap event per cell, or to simultaneously disrupt many independent genes in the same cell as a single provirus is inserted at each site. One of the disadvantages of retroviral delivery may be the presence of integration “hotspots” in the mouse genome, although this is not always observed (Hansen et al., 2003; Chen et al., 2004a). An advantage of plasmidbased gene trap vectors is that they are thought to integrate more randomly in the genome. However, they do not display an integration bias toward the 5⬘-end and, thus, integrate

more often than retroviral gene trap vectors along the length of a gene. This finding may result in a trap event that may not be as mutagenic, as functional partial proteins may be generated leading to a hypomorphic phenotype. Additionally, electroporation of the plasmid-based DNA gene trap vectors can lead to multiple or complex insertions in the genome, which can complicate the subsequent analysis of mutant mouse lines. The relatively high rates of embryonic lethality seen among mouse strains harboring homozygous null gene trap mutations has led to the development of gene trap vectors that allow for conditional gene inactivation, thus enabling studies of gene function at later stages of development and in the adult. Two groups have developed similar approaches that allow for conditional gene trapping with the use of site-specific recombinases (Schnu¨tgen et al., 2005; Xin et al., 2005). In the first of these approaches, a vector was engineered containing an inverted promoter trap cassette flanked by mutant loxP sites that are resistant to further inversion events and a bidirectional tandem polyA trap cassette flanked by FRT sites in direct orientation (Fig. 2A; Xin et al., 2005). After clonal growth, the polyA trap cassette can be removed by transiently expressing FLP recombinase. This process leaves the promoter trap cassette still present but in the opposite orientation with respect to the trapped gene, and thus silenced. Upon mating to a Cre-expressing mouse, the mutagenic gene inactivation cassette can be inverted, conditionally disrupting the gene. Similar vectors termed flip-excision (Flex; Schnu¨tgen et al., 2005) also have been created. These vectors feature sites for two different DNA recombinase systems placed in opposite orientation flanking a single mutagenic cassette (Fig. 2B; Schnu¨tgen et al., 2005). Upon recombination with FLP, the gene trap cassette is inverted with respect to the trapped gene and, therefore, silenced. To re-activate the gene trap cassette in a temporal or spatial manner, the mouse can be crossed to any Cre-expressing transgenic line. Moreover, this group has performed a large-scale conditional mutagenesis in ES cells using these vectors and has


gous recombination (Friedel et al., 2005). The authors showed that by flanking a promoterless secretory gene trap vector with standard 5⬘- and 3⬘-arms of homology, they achieved homologous targeting frequencies averaging better than 50%. This success is believed to be due to the majority of random insertions being eliminated by drug selection. The authors were able to target successfully 18 of 24 genes with this method. Of the six targeting attempts that failed, all genes were found to be expressed at low levels in ES cells, perhaps lower than the threshold limit needed to confer drug resistance. Although the “targeted trapping” described in this manuscript was performed with a promoterless secretory trap vector, this method could be applied readily to other promoterless gene trap vectors.

GENE TRAP SCREENS

Fig. 2. Schematic for conditional gene trapping. A: A conditional gene trap vector containing a promoter trap module and two polyA traps in tandem but opposite orientations inserts into an intron of a transcriptionally active gene. Upon recombination with FLPe, the gene trap cassette is removed, therefore restoring endogenous gene function. To facilitate the identification of embryonic stem (ES) clones in which the gene trap cassette has been removed, a thymidine kinase (TK) gene is included for negative selection. Upon further recombination with Cre, the gene inactivation cassette is inverted to the mutagenic orientation. loxLE is also known as lox71, and loxRE as lox66. B: A conditional gene trap vector featuring a SA␤geopA reporter cassette flanked by WT (loxP, FRT) and mutant (lox511, F3) recognition sequences for Cre and FLPe recombinase placed in opposite orientation. The gene trap is inverted and inactivated upon FLPe recombination. The mutagenic reporter cassette can be re-activated upon recombination with Cre.

generated a library of greater than 4,000 gene trap clones, representing insertions in 1,000 unique genes. These clones are made available from the German Gene trap Consortium (http://genetrap.gsf.de/). Although promoterless selection cassettes have been used in gene tar-

geting for many years (Doetschman et al., 1988), this approach recently has enjoyed new attention. Friedel et al. have demonstrated that the use of promoterless gene trap selection cassettes in gene targeting, termed “targeted trapping,” leads to a significant increase in the efficiency of homolo-

The first gene trap screen to identify developmentally required genes was carried out by Friedrich and Soriano (Friedrich and Soriano, 1991). The authors generated chimeric mouse lines from pools of mutant ES cells harboring promoter traps. The offspring of these chimeric mice were examined for lacZ staining at patterns various stages of embryonic development. The embryos displayed restricted to widespread staining patterns. Of 24 gene trap lines, 9 failed to generate viable homozygous mutant offspring, indicating that the disrupted genes are required for development. Consistent with these rates of embryonic lethality, a more recent gene trap screen of developmentally regulated genes revealed that nearly one third of the 60 mouse lines generated from the trapped ES cell clones leads to embryonic or postnatal lethality (Mitchell et al., 2001). Another large-scale screen characterized 279 expression patterns and identified a significant proportion of genes that are temporally or spatially regulated during embryogenesis (Wurst et al., 1995). Several gene trap screens have been carried out to identify genes involved in specific aspects of development. For example, in an effort to identify genes involved in mammalian axon guidance, a phenotype-driven modified gene trap screen was performed using


a PLAP secretory vector (Leighton et al., 2001). Secretory trap vectors include a modified ␤geo fused to a transmembrane domain causing the fusion protein to associate with the plasma membrane (Tashiro et al., 1993; Skarnes et al., 1995). The PLAP secretory vector features an additional human placental alkaline phosphatase (PLAP) reporter downstream of an IRES sequence. If a trapped gene is expressed in a neuron, the lacZ staining would only be detectable in the neuronal cell body. However, because PLAP is a glycosyl phosphatidylinositol (GPI) -linked cell surface marker, it allows for the complete labeling of axons, thus proving useful for the identification of mutant lines with abnormal axon guidance phenotypes. Differentiation gene trap screens in ES cells can be carried out to identify genes expressed in specific cell types. A recent example of such an ES cell screen was carried out to identify endothelial-specific genes (Hirashima et al., 2004). After generating a master library of 864 ES cell clones trapped with a PolyA trap vector, replicates were grown on an OP9 feeder cell layer, known to induce endothelial differentiation of ES cells (Kataoka et al., 1997). After 5 days of culture on these feeder cells, the authors identified five clones that displayed specific up-regulation of their gene trap lacZ reporter gene in response to the endothelial differentiation conditions. The authors successfully cloned three of the gene trap insertion by RACE. Of interest, two of the trapped genes were shown previously to be specifically expressed in endothelial cells, thus providing proof-of-principle for this approach. To identify and trap developmentally regulated genes that are expressed at low levels in ES cells, a bigenic approach was developed that uses a gene trap vector and expresses Cre upon insertion into an active gene and an ES cell line that carries a Cre reporter that confers drug resistance upon recombination (Chen et al., 2004b). ES cells may be treated with a stimulus such as retinoic acid, resulting in the induction of Cre expression from the trapped gene. Even if Cre is expressed at a low level from the trapped locus, it should be sufficient to induce recombination at the Cre re-

Fig. 3. ROSAFARY gene trap-coupled microarray analysis. Insertion of ROSAFARY gene trap vector into transcriptionally active gene. The 3⬘-rapid amplification of cDNA ends transcripts are generated from the polyA trap module and printed onto microarray chip and a duplicate library of embryonic stem (ES) cell clones harboring the ROSAFARY gene trap vector is frozen. The microarray chip can be used for transcriptional profiling experiments to identify genes of interest. Genetrapped ES cell lines with insertions in the genes of interest from the microarray experiments can be retrieved from frozen library stocks and mutant mouse lines generated.

porter locus, and confer a high-level of drug resistance. This bigenic system is extremely sensitive and may detect genes of interest that would normally go undetected with direct neomycin selection. Medico et al. developed a method for identifying transcriptionally responsive genes by fluorescence-activated cell sorting (FACS) analysis (Medico et al., 2001). The authors developed a retroviral gene trap vector featuring an enhanced green fluorescent protein fused to an Escherichia coli nitroreductase gene (GFNR) downstream of a splice acceptor sequence. Using positive or negative selection with metronidazole followed by FACS analysis, the authors were able to detect genes that were either transcriptionally upor down-regulated upon stimulation with hepatocyte growth factor. Although an embryonic mouse liver cell line was used in this screen, this technique could equally be applied to screening ES cells. Another recent development couples gene trap mutagenesis with microarray expression profiling allowing for the identification of transcriptionally responsive genes (Chen et al., 2004a). These authors used the ROSAFARY vector, which consists of a standard promoter trap vector in tan-

dem with a polyA trap vector to generate a library of trapped ES cell clones (Fig. 3; Chen et al., 2004a). The PolyA trap module was used to generate cDNA tags by 3⬘-RACE that were subsequently spotted onto a microarray chip. These chips can then be used to perform transcriptional profiling experiments on any physiologically relevant cell type. After identification of genes of interest, mutant mouse lines can be generated from the corresponding gene-trapped ES cell clone in the frozen library stock. This approach bypasses the time-consuming process in which a gene targeting construct would need to be generated to functionally validate the transcriptional target. The authors also demonstrate that this retroviral-based vector is highly mutagenic, as 83% of the gene trap insertions occurred in the 5⬘-untranslated region or the first half of the coding sequence, suggesting that they are likely to represent null alleles.

NEW RESOURCES FOR INSERTIONAL MUTAGENESIS In 2001, several academic, government, and private mouse research programs led an initiative calling for


Fig. 4. Schematic of Mutagenic Insertion and Chromosome Engineering Resource (MICER) vectors. Vectors carrying the 3⬘- or 5⬘-portion of an Hprt minigene, drug resistance cassettes (Neo or Puro), and minigenes for the coat color markers agouti (A) and tyrosinase (Tyr) are shown. Insertion of these vectors into a gene may lead either to a frameshift mutation or a splicing mutation.

the formation of the International Mouse Mutagenesis Consortium (IMMC) with the goal of assigning function to every gene in the genome, and of identifying genes of high biomedical interests (Nadeau et al., 2001). Among the goals of the IMMC is the production of at least one heritable mutation, in either ES cells or mice, in every gene in the mouse genome. Moreover, the IMMC seeks to develop the infrastructure for archiving and distributing mutant ES cells and mice. In their initial proposal, the group suggested the use of both chemical and insertional mutagens, however, more recent initiatives have favored the latter. The Knockout Mouse Project (KOMP; http://www.nih.gov/ science/models/mouse/knockout/) was also formed recently with the goal of implementing a genome-wide mouse mutagenesis program (Austin et al., 2004). This group argued for the generation of null-reporter alleles, in contrast to conditional alleles, because it was believed that current technology did not allow for conditional mutagenesis in a cost-effective manner (Austin et al., 2004). However, it is estimated that one quarter to one third of homozygous null mutations are embry-

onic lethal, and direct gene trapping at such loci would preclude the study of gene function at later stages in development (Friedrich and Soriano, 1991; Mitchell et al., 2001). The European Conditional Mouse Mutagenesis Program (EUCOMM), which also recently was formed with similar goals to that of KOMP (Auwerx et al., 2004), emphasizes the development of new mutational strategies for conditional reporter alleles in the mouse. In addition, a third initiative, the North American Conditional Mouse Mutagenesis Project (NorCOMM), recently was funded by Genome Canada. Although this North American initiative only includes Canada, it has partnered with EUCOMM to produce large-scale libraries of gene trap or gene targeted ES cell clones to be made freely available to the academic community. Two large-scale efforts have been conducted to generate ES cell clones with a gene trap in every gene. Lexicon Genetics has developed Omnibank, the largest collection of mutant ES cells. This library consists of over 270,000 clones with an approximate 60% coverage of the genome (Zambrowicz et al., 2003). Approximately

200,000 sequence tags of the commercially available Omnibank ES cell gene trap clones have been deposited into the NCBI Genome Survey Sequences Database (dbGSS). A searchable database of this library is also provided at the company’s Web site (http://www.lexicon-genetics.com/ index.php). The large drawbacks to this resource are the relative highcost to academic researchers, limits on intellectual property rights, and that not all of the clones are made available for public purchase. A similar effort has been initiated by the academic community under the International Gene Trap Consortium (IGTC) with the goal of providing a free, publicly available ES cell gene trap library (Skarnes et al., 2004). The IGTC already has generated a collection of 50,000 ES cell lines. Although this number is a lower total number of ES lines than Omnibank, the IGTC library represents mutation in 9,000 known mouse genes, or approximately 40% of known mouse genes. Of interest, the IGTC’s use of a diverse set of gene trap vectors has trapped a larger number of distinct loci in a fewer number of clones compared with Lexicon’s Omnibank (Skarnes et al., 2004). The publicly available trapped sequence tags are archived in a fully annotated and searchable database at the IGTC Web site (http://www.genetrap.org/; Nord et al., 2006). The clones may also be visualized with the Ensembl mouse genome browser (http://www. ensembl.org/Mus_musculus/index.html) under the DAS source Gene Trap. Another recent technological development to help facilitate the analysis of mammalian gene function is the Mutagenic Insertion and Chromosome Engineering Resource (MICER; Adams et al., 2004). MICER consists of two publicly available libraries containing of 93,960 ready-made insertional targeting vectors. The 5⬘-Hprt (MHPN) and 3⬘-Hprt (MHPP) insertional targeting vector libraries consist of a drug resistance cassette, the 5⬘- or 3⬘-part of an Hprt-minigene, a loxP site for chromosome engineering, and a mouse coat-color marker for ease of maintenance of the targeted mouse strain, and regions of homology to the mouse genome (Fig. 4; Adams et al., 2004). Although de-


signed to be used in pairs to create large-scale deletions, duplications, translocations, or inversions, these insertion vectors can be used alone to inactivate genes through homologous recombination in ES cells. Upon insertion of the MICER vectors, the region of homology undergoes a duplication that is usually sufficient to create a frameshift mutation due to the extra sequence. Approximately 11.2% of mouse genes are readily accessible for disruption with the current vector libraries. This targeting vector resource is available publicly through the MICER Web site (http://www.sanger. ac.uk/PostGenomics/mousegenomics/), and the insertion sites are mapped on the Ensembl mouse genome browser Web site (http://www.ensembl.org/Mus_ musculus/index.html) under the DAS source MICER. On average, a targeting efficiency of 28% has been observed with these targeting vectors. This resource will prove to be even more valuable as additional libraries are constructed. Recent advances in technology have allowed the development of bacterial artificial chromosome (BAC) -based gene targeting mutagenesis in the mouse (Testa et al., 2003; Valenzuela et al., 2003). Regeneron Pharmaceuticals has developed VelociGene, an automated high-throughput method using targeting vectors to generate deletions in the mouse genome (Fig. 5; Valenzuela et al., 2003). The VelociGene system replaces the genomic locus with a reporter gene that allows for the analysis of expression patterns in targeted genes. In this approach, short arms of homology (usually 50 – 200 bp) matching the target site of insertion are generated from doublestranded oligonucleotides that are ligated to the ends of a tandem-linked reporter and selection cassette. This reporter-selection cassette is then introduced into recombinogenic bacterial strains to undergo homologous recombination with a BAC containing the gene of interest creating a targeting vector, or BACvec. This BACvec is then linearized and electroporated into ES cells and is assayed using real-time quantitative PCR to identify ES cells that have undergone homologous recombination by screening for a “loss-of-native” allele. Using this sys-

Fig. 5. Regeneron Velocigene System. Bacterial artificial chromosome (BAC) clones containing a DNA region of interest to be deleted are identified by polymerase chain reaction (PCR). The 50 to 200-bp arms of upstream (uHOM) and downstream (dHOM) homology that define the region to be deleted are generated and ligated to a selection/reporter cassette. This reporter cassette vector and BAC clone undergo a recombineering process to generate a targeted BACvec. This BACvec can then be electroporated into embryonic stem (ES) cells where an automated Q-PCR “loss of native” allele analysis can reveal successfully targeted clones.

tem, the authors generated deletions in the mouse genome up to 70 kb with an average targeting efficiency of 3.8%. Due to the very large arms of homology on the BACvecs, they appear to not require isogenicity with the ES cell DNA, therefore, allowing the use of BAC clones and ES cell lines of different genetic backgrounds.

TRANSPOSON-MEDIATED MUTAGENESIS IN VERTEBRATES Transposable elements have been important tools for genetic manipulation; however, their use has been restricted to plants and lower metazoic organisms such as worms and flies. In the nematode C. elegans and Drosophila, Tc1 and P elements have proven useful for analysis of gene function. Two types of mobile genetic elements have been identified in the mouse: DNA transposons and retrotransposons. Both of these elements are mobilized by a “cut and paste” mechanism. DNA transposons are excised from their donor locus and re-inte-

grated at a different location in a process mediated by the transposase enzyme (Fig. 6). Retrotransposons are first transcribed and then reverse transcribed by the element-encoded enzymes resulting in the transposition of the genetic element to a new position in the genome. The use of transposon-based mutagenesis systems in the mouse recently has emerged with the resurrection of the first mobile vertebrate transposon, Sleeping Beauty (SB; Ivics et al., 1997). SB is a synthetic transposon and a member of the Tc1/Mariner family of transposable elements (for review, see Izsvak and Ivics, 2004). SB has been shown to be active in mouse and human cells, as well as the mouse germline (Ivics et al., 1997; Luo et al., 1998; Yant et al., 2000; Dupuy et al., 2001; Fischer et al., 2001; Horie et al., 2001; Carlson et al., 2003). SB is a binary DNA transposon system that consists of a transposase enzyme and a mobile transposable genetic element. The transposon element consists of a sequence of DNA flanked by inverted repeat (IR)/direct repeat ele-


Fig. 6. Schematic of vertebrate transposon system. The transposase enzyme recognizes inverted terminal repeats (ITR) of vertebrate transposable element, resulting in mobilization of transposon by a “cut and paste” mechanism. DNA undergoes repair at the donor site of transposition, and transposon inserts at the target-site recognition sequence at the new genomic locus.

ments. The transposase recognizes the IR elements excising them from the donor site and leaving a canonical three base pair footprint [C(A/T)G]. The mobilized element inserts at a new genomic locus at a random TA dinucleotide that is duplicated upon insertion. SB transposition, however, seems to be subject to a “local hopping” effect where the new site of integration is closely and physically linked to the donor site. This observation has been found to occur in approximately 50 – 80% of the transposition events (Carlson et al., 2003; Horie et al., 2003). Whereas this finding may limit genome-wide mutagenesis screens, one advantage of this local hopping phenomenon is that it may facilitate saturation mutagenesis near the donor site of transposition (Keng et al., 2005). However, recent studies have overcome these limitations with the development of a more efficient transposable element sequence and the continuous and ubiquitous expression of the transposase from the ROSA26 locus (Dupuy et al., 2005). This study showed an enhanced effect of creating germline mutations with nearly one quarter of double transgenic animals resulting in lethality. The real strength of the SB transposon system, however, is its ability to act as an insertional mutagen in somatic cells. It has been shown that SB transposonmediated mutagenesis generated a high-frequency of aggressive tumors in

the mouse (Collier et al., 2005; Dupuy et al., 2005). Genetic screens using the SB transposon system should facilitate the identification of genes involved in both cancer and development. Another vertebrate transposon, Minos, has also been described recently (Klinakis et al., 2000; Zagoraiou et al., 2001; Drabek et al., 2003). Minos, like SB, is a member of the Tc1/Mariner transposon family. Originally isolated from Drosophila hydei, Minos demonstrated the ability to act as a mobile genetic element in several insect species (for review see Handler, 2001). Minos also was found to be highly active in cultured human HeLa cells, suggesting its use for mammalian mutagenesis (Klinakis et al., 2000). Like other members of the Tc1/Mariner superfamily of transposons, Minos recognizes and integrates at a TA dinucleotide that is duplicated upon excision. Transgenic mice carrying the Minos transposase cDNA under the control of the human CD2 promoter mobilized a Minos transposon carrying a CMVGFP reporter gene in a CD2-specific manner (Zagoraiou et al., 2001). However, the efficiency of transposition was lower in mouse T cells than in HeLa cells and also lower than that reported for SB in mouse embryonic stem cells. Using a zona pellucida (ZP3) -driven Minos transposase transgenic mouse line, Drabek et al. demonstrated transposition of

the Minos element in the female germline in 8.2% of the double transgenic progeny (Drabek et al., 2003). Moreover, they found that, among 11 transposition events analyzed, 8 had mobilized to a different chromosome, suggesting little “local hopping.” Another promising vertebrate transposon system, the DNA transposon piggyBac (PB) originally identified from the cabbage looper moth Trichoplusia ni, was shown to transpose in more than a dozen insect species, as well as in human and mouse cell lines and in the germline of mice (Ding et al., 2005). The PB transposable element includes a 594 amino acid transposase flanked by 13-bp inverted terminal repeats (ITRs). The PB transposons recognize and insert at the tetranucleotide sequence TTAA, which is duplicated upon insertion. Like other transposon systems mentioned above, PB inserts as a stable single copy insertion and the transposon tag allows for easy identification and cloning of the transgene insertion site. The PB system allows for expression of transgenes up to 14.3 kb in length; however, the transposition frequency decreases with larger transgenes. PB appears to integrate randomly throughout the mouse genome, but two thirds of the analyzed insertions occurred within known or predicted transcriptional units. PB also leaves no footprint after excision allowing for phenotypic reversion of the mutation back to the wild-type allele, providing the investigator with the opportunity to verify the nature of the relationship between the mutation and phenotype. An active human-derived LINE-1 (L1), a member of the LINE-1 family of retrotransposons, was shown to mobilize in cultured human cells as well as in the mouse germline (Moran et al., 1996; Ostertag et al., 2002). However, the low frequency of such germline transposition events (around 1%) is probably not sufficient for this to be used as a germline mutagen. Recent increases in transposition frequency up to 200-fold were observed upon reengineering the L1 open reading frame with synthetic codons (Han and Boeke, 2004). These improvements show promise for using this system as an in vivo insertional mutagen. However, two drawbacks to this system


are that L1 vectors can be truncated at the 5⬘-end and cause deletions at the insertion sites (Gilbert et al., 2002; Farley et al., 2004).

PERSPECTIVES With new mutational initiatives in place, large-scale screens currently under way, and the resulting resources openly available to the academic community, the coming years should enable researchers to accelerate functional analysis of genes involved in vertebrate development and disease. The challenges in the field of insertional mutagenesis will lie mostly in the advancement of technology, as well as improving vector design and screening strategies to generate more efficient tools for insertional mutagenesis. For instance, vectors will need to be designed to carry out specialized screens, such as gainof-function or sensitized-background screens, to identify genes involved in specific developmental processes. There are several challenges for the large-scale mutagenesis screens that are now under way under the directives of KOMP, EUCOMM, and NorCOMM. One issue is to define the best genetic background for performing these screens, either C57BL6 or 129. KOMP currently favors performing these screens with C57BL6 mice, as this is the genetic background on which the initial sequence of the mouse genome is derived and is commonly used, especially in immunology. However, one of the main disadvantages of using this genetic background is that it is more difficult to generate C57BL6-derived ES cell lines de novo. In addition, C57BL6derived ES cells have not been used frequently and never in a highthroughput capacity, so it is not known how efficient these cells are at generating high-percentage germline chimeras. More work will be needed to develop technology that will improve the efficiency of generating C57BL6based ES cell lines de novo, or technology that would allow for the efficient use of current C57BL6 ES cell lines. EUCOMM currently takes a different stand on this issue by favoring the use of ES cells from the more common and efficient 129 genetic background. EUCOMM stresses that the efforts of the

public gene trap consortium have already generated gene trap mutations in nearly 40% of the known genes in the genome. Moreover, the conditional mutagenesis approach favored by this entity is more conducive and compatible with the technology used to generate these conditional alleles. Alternatively, the groups could opt to use of ES cells of a hybrid 129/C57BL6 F1 genetic background. Such ES cell lines have been shown to be very efficient in generating germline chimeras. These cells display a “hybrid vigor” that is also useful in generating completely ES cell-derived lines by tetraploid complementation and nuclear cloning approaches (Rideout et al., 2000; Eggan et al., 2001). However, the downside of using an ES cell line of a mixed genetic background, even though they are very efficient, is the added problems with phenotypic analysis of the mutant lines. The complex genetic backgrounds could reveal or mask modifiers of phenotypes that may not be present if the mutant lines were carried on a homogenous genetic background. Ideally, the choice between these two genetic backgrounds would not be an either/or situation. It may be beneficial if both entities developed complementary approaches by using ES cells of different genetic background, thus leading to the potential generation of mutant alleles on both. This approach would highlight genetic modifiers if one mutation presented a phenotype that was not present on the other background, and could facilitate further modifier screens to elucidate various genetic and developmental pathways. Another question that arises for these large-scale mutagenesis projects is what is the best reporter system to use with the various insertional mutagenesis techniques. The two classic options for a reporter gene are either lacZ or green fluorescent protein (GFP). LacZ has the advantage that it is a rather straightforward histochemical stain that requires no special equipment to reveal high-resolution staining patterns in young embryonic fixed whole-mount embryos. The disadvantage of this histological marker is the low penetration of the X-Gal stain into the embryo or tissue, thus making it only useful to reveal structures, tissues, or cell types near the

surface. Older embryos and adult organs must first be vibrio-sectioned before staining can be performed. Another disadvantage of using lacZ as a marker is that staining can only be performed on fixed tissue, therefore, not allowing for the monitoring of gene expression in living ES cells or embryos. The use of GFP as a reporter marker is compatible with imaging gene expression patterns in living animals as well as cell culture, but the disadvantage of this system is that it requires special equipment to visualize and does not easily provide a permanent high-resolution image of the gene expression pattern. The recent development of a nontoxic monomeric form of the red fluorescent protein DsRed (mRFP1) allows for the use of an alternative fluorescent reporter for imaging gene expression in mouse embryos and ES cells (Long et al., 2005; Zhu et al., 2005). This reporter appears to be much more sensitive than GFP, while preserving all of its advantages. After the generation of all of these mutations by the large-scale screens, either in ES cells or mutant mouse lines, comes the challenge of where and how to store them for future analysis. Recently, the mouse repositories of North America, Europe, and Asia have collaborated to form the Federation of International Mouse Resources (FIMRe) (http://www.fimre.org), an organization focused on coordinating mouse archival resources for the scientific community. Currently, however, this collaboration appears to be still in its infancy as each of these repositories still functions independently from one another, with no central database to access information on mutant mouse strains and cell lines. Further coordination between these international repositories will be needed to efficiently catalog, store, and distribute this material. Another question remains as to what is the best form in which to store these mutant alleles. As of now, the options are few. Live colonies of mice could be maintained, which would have the benefit of mouse lines readily available for phenotypic analysis. However, the costs of maintaining large colonies of mice by a repository are prohibitive. ES cells, sperm, or embryos can all be cryopreserved as a


TABLE 1. Mouse Informatics Resources Gene trap International Gene Trap Consortium (IGTC) Baygenomics Sanger Institute Gene Trap Resource (SIGTR) Mammalian Functional Genomics Centre (MFGC) German Gene Trap Consortium (GGTC) Centre for Modeling Human Disease (CMHD) Soriano Gene Trap Database

http://www.genetrap.org/ http://baygenomics.ucsf.edu/index.html http://www.sanger.ac.uk/PostGenomics/genetrap/ http://escells.ca/ http://genetrap.gsf.de/ http://www.cmhd.ca/genetrap/index.html http://fhcrc.org/science/labs/soriano/trap.html

Repositories Federation of International Mouse Resources The Jackson Laboratory Mutant Mouse Regional Resource Centers (MMRRC) European Mouse Mutant Archive (EMMA)

http://www.fimre.org/ http://www.jax.org/ http://www.mmrrc.org/ http://www.emma.rm.cnr.it/

Informatics Ensembl Mouse Mouse Genome Informatics (MGI) Insertional Mouse Strain Resource (IMSR) Mouse Phenome Database (MPD) Gene Expression Database (GXD) Trans-NIH Mouse Initiatives

http://www.ensembl.org/Mus_musculus/index.html http://www.informatics.jax.org/ http://www.informatics.jax.org/imsr/index.jsp http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn⫽docs/home http://www.informatics.jax.org/menus/expression_menu.shtml http://www.nih.gov/science/models/mouse/resources/index.html

low-cost alternative to maintaining living colonies of mice. However, the cryopreservation techniques involved with both the freezing of sperm and embryos are difficult and are often associated with high mortality. Therefore, if this is to be the method of choice for reliable long-term systematic storage mutant mouse embryos or sperm, additional advances in cryopreservation techniques will be needed. ES cells, on the other hand can be readily frozen and thawed reliably but require the further generation of chimeras by blastocyst injection or morula aggregation to generate mouse lines. With the generation of libraries of mutant ES cells and ready-made vectors for gene targeting every gene in the genome, we will face the additional challenges of archiving and distributing all of this information so that researchers have ready access to the data. Several Web-based resources are already in place that provides access to this information (Table 1). However, they are not always well publicized, and although some of these databases are cross-referenced to one another, many are not. Therefore, an investigator often needs to laboriously search each site to extract the needed information. A central Web-based informatics database will need to be established that will serve as a bank of information into which all

mutant alleles will be cataloged, including any known phenotypes and expression patterns, as well as providing accessibility to obtaining these mutant lines. Additionally, the mouse nomenclature system currently in place does not provide a clear description of the nature of the mutant allele (http://www.informatics.jax.org/mgihome/nomen/gene.shtml). The current system for assigning an allele designation is based on the laboratory in which the mutation was generated and not on the description of the nature of the mutation itself. This approach often leads to confusion among investigators when trying to obtain specific mutant alleles. A revision of the mouse nomenclature system in the future will help facilitate access for investigators to the desired mutant strains. Despite these hurdles, all of the ongoing efforts and high resolution technologies in mouse insertional mutagenesis should increase the speed in which we will able to dissect genetic pathways, understand developmental processes, and relate gene function to human disease.

ACKNOWLEDGMENTS We apologize to many colleagues whose work we could not cite due to space limitations. We thank our laboratory colleagues for critical comments on the manuscript. C.R. was

supported by a fellowship from the Leukemia & Lymphoma Society and a Chromosome Metabolism and Cancer training grant fellowship from the National Institutes of Health. Work in the author’s laboratory is supported by grants from the National Institute of Child Health and Human Development to P.S.

REFERENCES Adams DJ, Biggs PJ, Cox T, Davies R, van der Weyden L, Jonkers J, Smith J, Plumb B, Taylor R, Nishijima I, Yu Y, Rogers J, Bradley A. 2004. Mutagenic insertion and chromosome engineering resource (MICER). Nat Genet 36:867– 871. Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT, Grieder FB, Heintz N, Hicks G, Insel TR, Joyner A, Koller BH, Lloyd KC, Magnuson T, Moore MW, Nagy A, Pollock JD, Roses AD, Sands AT, Seed B, Skarnes WC, Snoddy J, Soriano P, Stewart DJ, Stewart F, Stillman B, Varmus H, Varticovski L, Verma IM, Vogt TF, von Melchner H, Witkowski J, Woychik RP, Wurst W, Yancopoulos GD, Young SG, Zambrowicz B. 2004. The knockout mouse project. Nat Genet 36:921–924. Auwerx J, Avner P, Baldock R, Ballabio A, Balling R, Barbacid M, Berns A, Bradley A, Brown S, Carmeliet P, Chambon P, Cox R, Davidson D, Davies K, Duboule D, Forejt J, Granucci F, Hastie N, de Angelis MH, Jackson I, Kioussis D, Kollias G, Lathrop M, Lendahl U, Malumbres M, von Melchner H, Muller W, Partanen J, Ricciardi-Castagnoli P, Rigby P,


Rosen B, Rosenthal N, Skarnes B, Stewart AF, Thornton J, Tocchini-Valentini G, Wagner E, Wahli W, Wurst W. 2004. The European dimension for the mouse genome mutagenesis program. Nat Genet 36:925–927. Carlson CM, Dupuy AJ, Fritz S, RobergPerez KJ, Fletcher CF, Largaespada DA. 2003. Transposon mutagenesis of the mouse germline. Genetics 165:243–256. Caspary T, Anderson K. 2006. Uncovering the uncharacterized and unexpected: unbiased, phenotype driven screens in the mouse. Dev Dyn 235:2412–2423. Chen WV, Soriano P. 2003. Gene trap mutagenesis in embryonic stem cells. Methods Enzymol 365:367–386. Chen Y, Yee D, Dains K, Chatterjee A, Cavalcoli J, Schneider E, Om J, Woychik RP, Magnuson T. 2000. Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nat Genet 24:314 –317. Chen WV, Delrow J, Corrin PD, Frazier JP, Soriano P. 2004a. Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis. Nat Genet 36:304 –312. Chen YT, Liu P, Bradley A. 2004b. Inducible gene trapping with drug-selectable markers and Cre/loxP to identify developmentally regulated genes. Mol Cell Biol 24:9930 –9941. Collier LS, Carlson CM, Ravimohan S, Dupuy AJ, Largaespada DA. 2005. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436:272–276. Copeland NG, Jenkins NA, Court DL. 2001. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769 –779. De-Zolt S, Schnutgen F, Seisenberger C, Hansen J, Hollatz M, Floss T, Ruiz P, Wurst W, von Melchner H. 2006. Highthroughput trapping of secretory pathway genes in mouse embryonic stem cells. Nucleic Acids Res 34:e25. Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T. 2005. Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122:473–483. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. 1987. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576 –578. Doetschman T, Maeda N, Smithies O. 1988. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci U S A 85:8583–8587. Drabek D, Zagoraiou L, deWit T, Langeveld A, Roumpaki C, Mamalaki C, Savakis C, Grosveld F. 2003. Transposition of the Drosophila hydei Minos transposon in the mouse germ line. Genomics 81: 108 –111. Dupuy AJ, Fritz S, Largaespada DA. 2001. Transposition and gene disruption in the male germline of the mouse. Genesis 30: 82–88. Dupuy AJ, Akagi K, Largaespada DA, Copeland NG, Jenkins NA. 2005. Mammalian mutagenesis using a highly mo-

bile somatic Sleeping Beauty transposon system. Nature 436:221–226. Eggan K, Akutsu H, Loring J, JacksonGrusby L, Klemm M, Rideout WM III, Yanagimachi R, Jaenisch R. 2001. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 98:6209 –6214. Farley AH, Luning Prak ET, Kazazian HH Jr. 2004. More active human L1 retrotransposons produce longer insertions. Nucleic Acids Res 32:502–510. Fischer SE, Wienholds E, Plasterk RH. 2001. Regulated transposition of a fish transposon in the mouse germ line. Proc Natl Acad Sci U S A 98:6759 –6764. Friedel RH, Plump A, Lu X, Spilker K, Jolicoeur C, Wong K, Venkatesh TR, Yaron A, Hynes M, Chen B, Okada A, McConnell SK, Rayburn H, TessierLavigne M. 2005. Gene targeting using a promoterless gene trap vector (“targeted trapping”) is an efficient method to mutate a large fraction of genes. Proc Natl Acad Sci U S A 102:13188 –13193. Friedrich G, Soriano P. 1991. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513– 1523. Friedrich G, Soriano P. 1993. Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells. Methods Enzymol 225:681–701. Frohman MA. 1993. Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol 218: 340 –356. Gilbert N, Lutz-Prigge S, Moran JV. 2002. Genomic deletions created upon LINE-1 retrotransposition. Cell 110:315–325. Gossler A, Joyner AL, Rossant J, Skarnes WC. 1989. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science 244: 463–465. Green EL, Roderick TH. 1966. Radiation genetics. New York: McGraw-Hill. Han JS, Boeke JD. 2004. A highly active synthetic mammalian retrotransposon. Nature 429:314 –318. Handler AM. 2001. A current perspective on insect gene transformation. Insect Biochem Mol Biol 31:111–128. Hansen J, Floss T, Van Sloun P, Fuchtbauer EM, Vauti F, Arnold HH, Schnutgen F, Wurst W, von Melchner H, Ruiz P. 2003. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A 100:9918 –9922. Hicks GG, Shi EG, Li XM, Li CH, Pawlak M, Ruley HE. 1997. Functional genomics in mice by tagged sequence mutagenesis. Nat Genet 16:338 –344. Hirashima M, Bernstein A, Stanford WL, Rossant J. 2004. Gene-trap expression screening to identify endothelial-specific genes. Blood 104:711–718. Horie K, Kuroiwa A, Ikawa M, Okabe M, Kondoh G, Matsuda Y, Takeda J. 2001. Efficient chromosomal transposition of a

Tc1/mariner-like transposon Sleeping Beauty in mice. Proc Natl Acad Sci U S A 98:9191–9196. Horie K, Yusa K, Yae K, Odajima J, Fischer SE, Keng VW, Hayakawa T, Mizuno S, Kondoh G, Ijiri T, Matsuda Y, Plasterk RH, Takeda J. 2003. Characterization of Sleeping Beauty transposition and its application to genetic screening in mice. Mol Cell Biol 23:9189 –9207. Hrabe de Angelis MH, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, Marschall S, Heffner S, Pargent W, Wuensch K, Jung M, Reis A, Richter T, Alessandrini F, Jakob T, Fuchs E, Kolb H, Kremmer E, Schaeble K, Rollinski B, Roscher A, Peters C, Meitinger T, Strom T, Steckler T, Holsboer F, Klopstock T, Gekeler F, Schindewolf C, Jung T, Avraham K, Behrendt H, Ring J, Zimmer A, Schughart K, Pfeffer K, Wolf E, Balling R. 2000. Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 25:444 –447. Ivics Z, Hackett PB, Plasterk RH, Izsvak Z. 1997. Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91:501–510. Izsvak Z, Ivics Z. 2004. Sleeping beauty transposition: biology and applications for molecular therapy. Mol Ther 9:147– 156. Jenkins NA, Copeland NG. 1985. High frequency germline acquisition of ecotropic MuLV proviruses in SWR/J-RF/J hybrid mice. Cell 43:811–819. Kataoka H, Takakura N, Nishikawa S, Tsuchida K, Kodama H, Kunisada T, Risau W, Kita T, Nishikawa SI. 1997. Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells. Dev Growth Differ 39:729 –740. Keng VW, Yae K, Hayakawa T, Mizuno S, Uno Y, Yusa K, Kokubu C, Kinoshita T, Akagi K, Jenkins NA, Copeland NG, Horie K, Takeda J. 2005. Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods 2:763–769. Kile BT, Hentges KE, Clark AT, Nakamura H, Salinger AP, Liu B, Box N, Stockton DW, Johnson RL, Behringer RR, Bradley A, Justice MJ. 2003. Functional genetic analysis of mouse chromosome 11. Nature 425:81–86. Klinakis AG, Zagoraiou L, Vassilatis DK, Savakis C. 2000. Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos. EMBO Rep 1:416 –421. Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skarnes WC, Tessier-Lavigne M. 2001. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410:174 –179. Long JZ, Lackan CS, Hadjantonakis AK. 2005. Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a


monomeric red fluorescent protein. BMC Biotechnol 5:20. Luo G, Ivics Z, Izsvak Z, Bradley A. 1998. Chromosomal transposition of a Tc1/ mariner-like element in mouse embryonic stem cells. Proc Natl Acad Sci U S A 95:10769 –10773. Medico E, Gambarotta G, Gentile A, Comoglio PM, Soriano P. 2001. A gene trap vector system for identifying transcriptionally responsive genes. Nat Biotechnol 19:579 –582. Mitchell KJ, Pinson KI, Kelly OG, Brennan J, Zupicich J, Scherz P, Leighton PA, Goodrich LV, Lu X, Avery BJ, Tate P, Dill K, Pangilinan E, Wakenight P, Tessier-Lavigne M, Skarnes WC. 2001. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat Genet 28:241– 249. Moran JV, Holmes SE, Naas TP, DeBerardinis RJ, Boeke JD, Kazazian HH Jr. 1996. High frequency retrotransposition in cultured mammalian cells. Cell 87:917– 927. Munroe RJ, Bergstrom RA, Zheng QY, Libby B, Smith R, John SW, Schimenti KJ, Browning VL, Schimenti JC. 2000. Mouse mutants from chemically mutagenized embryonic stem cells. Nat Genet 24:318 –321. Nadeau JH, Balling R, Barsh G, Beier D, Brown SD, Bucan M, Camper S, Carlson G, Copeland N, Eppig J, Fletcher C, Frankel WN, Ganten D, Goldowitz D, Goodnow C, Guenet JL, Hicks G, Hrabe de Angelis M, Jackson I, Jacob HJ, Jenkins N, Johnson D, Justice M, Kay S, Kingsley D, Lehrach H, Magnuson T, Meisler M, Poustka A, Rinchik EM, Rossant J, Russell LB, Schimenti J, Shiroishi T, Skarnes WC, Soriano P, Stanford W, Takahashi JS, Wurst W, Zimmer A. 2001. Sequence interpretation. Functional annotation of mouse genome sequences. Science 291:1251–1255. Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K. 1993. An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events. J Biochem (Tokyo) 113: 343–349. Nolan PM, Peters J, Vizor L, Strivens M, Washbourne R, Hough T, Wells C, Glenister P, Thornton C, Martin J, Fisher E, Rogers D, Hagan J, Reavill C, Gray I, Wood J, Spurr N, Browne M, Rastan S, Hunter J, Brown SD. 2000. Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource. Mamm Genome 11:500 –506. Nord AS, Chang PJ, Conklin BR, Cox AV, Harper CA, Hicks GG, Huang CC, Johns SJ, Kawamoto M, Liu S, Meng EC, Morris JH, Rossant J, Ruiz P, Skarnes WC, Soriano P, Stanford WL, Stryke D, von Melchner H, Wurst W, Yamamura K, Young SG, Babbitt PC, Ferrin TE. 2006. The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res 34:D642–D648.

Ostertag EM, DeBerardinis RJ, Goodier JL, Zhang Y, Yang N, Gerton GL, Kazazian HH Jr. 2002. A mouse model of human L1 retrotransposition. Nat Genet 32:655–660. Popp RA, Bailiff EG, Skow LC, Johnson FM, Lewis SE. 1983. Analysis of a mouse alpha-globin gene mutation induced by ethylnitrosourea. Genetics 105:157–167. Rideout WM III, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L, Dausman J, Yanagimachi R, Jaenisch R. 2000. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nat Genet 24:109 –110. Rinchik EM. 1991. Chemical mutagenesis and fine-structure functional analysis of the mouse genome. Trends Genet 7:15– 21. Robertson E, Bradley A, Kuehn M, Evans M. 1986. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323:445–448. Russell WL, Kelly EM, Hunsicker PR, Bangham JW, Maddux SC, Phipps EL. 1979. Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc Natl Acad Sci U S A 76:5818 –5819. Russell LB, Hunsicker PR, Cacheiro NL, Bangham JW, Russell WL, Shelby MD. 1989. Chlorambucil effectively induces deletion mutations in mouse germ cells. Proc Natl Acad Sci U S A 86:3704 –3708. Schnieke A, Harbers K, Jaenisch R. 1983. Embryonic lethal mutation in mice induced by retrovirus insertion into the alpha 1(I) collagen gene. Nature 304:315– 320. Schnu¨tgen F, De-Zolt S, Van Sloun P, Hollatz M, Floss T, Hansen J, Altschmied J, Seisenberger C, Ghyselinck NB, Ruiz P, Chambon P, Wurst W, von Melchner H. 2005. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A 102:7221–7226. Shigeoka T, Kawaichi M, Ishida Y. 2005. Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells. Nucleic Acids Res 33:e20. Skarnes WC, Auerbach BA, Joyner AL. 1992. A gene trap approach in mouse embryonic stem cells: the lacZ reported is activated by splicing, reflects endogenous gene expression, and is mutagenic in mice. Genes Dev 6:903–918. Skarnes WC, Moss JE, Hurtley SM, Beddington RS. 1995. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc Natl Acad Sci U S A 92:6592–6596. Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, Conklin BR, Stanford WL, Rossant J. 2004. A public gene trap resource for mouse functional genomics. Nat Genet 36:543–544. Spence SE, Gilbert DJ, Swing DA, Copeland NG, Jenkins NA. 1989. Spontaneous germ line virus infection and retro-

viral insertional mutagenesis in eighteen transgenic Srev lines of mice. Mol Cell Biol 9:177–184. Stanford WL, Cohn JB, Cordes SP. 2001. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2:756 –768. Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. 1993. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600 –603. Testa G, Zhang Y, Vintersten K, Benes V, Pijnappel WW, Chambers I, Smith AJ, Smith AG, Stewart AF. 2003. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21:443–447. Thomas KR, Capecchi MR. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512. Usman N, Tarabykin V, Gruss P. 2000. The novel PCR-based technique of genotyping applied to identification of scrambler mutation in mice. Brain Res Brain Res Protoc 5:243–247. Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, Chernomorsky R, Boucher M, Elsasser AL, Esau L, Zheng J, Griffiths JA, Wang X, Su H, Xue Y, Dominguez MG, Noguera I, Torres R, Macdonald LE, Stewart AF, DeChiara TM, Yancopoulos GD. 2003. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21:652–659. Vivian JL, Chen Y, Yee D, Schneider E, Magnuson T. 2002. An allelic series of mutations in Smad2 and Smad4 identified in a genotype-based screen of N-ethyl-N- nitrosourea-mutagenized mouse embryonic stem cells. Proc Natl Acad Sci U S A 99:15542–15547. von Melchner H, DeGregori JV, Rayburn H, Reddy S, Friedel C, Ruley HE. 1992. Selective disruption of genes expressed in totipotent embryonal stem cells. Genes Dev 6:919 –927. Wagner EF, Covarrubias L, Stewart TA, Mintz B. 1983. Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line. Cell 35:647–655. Wu X, Li Y, Crise B, Burgess SM. 2003. Transcription start regions in the human genome are favored targets for MLV integration. Science 300:1749 –1751. Wurst W, Rossant J, Prideaux V, Kownacka M, Joyner A, Hill DP, Guillemot F, Gasca S, Cado D, Auerbach A, et al. 1995. A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice. Genetics 139:889 –899. Xin HB, Deng KY, Shui B, Qu S, Sun Q, Lee J, Greene KS, Wilson J, Yu Y, Feldman M, Kotlikoff MI. 2005. Gene trap and gene inversion methods for conditional gene inactivation in the mouse. Nucleic Acids Res 33:e14. Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay MA. 2000. Somatic integration and long-term transgene expression in normal


and haemophilic mice using a DNA transposon system. Nat Genet 25:35–41. Yoshida M, Yagi T, Furuta Y, Takayanagi K, Kominami R, Takeda N, Tokunaga T, Chiba J, Ikawa Y, Aizawa S. 1995. A new strategy of gene trapping in ES cells using 3⬘RACE. Transgenic Res 4:277–287. Zagoraiou L, Drabek D, Alexaki S, Guy JA, Klinakis AG, Langeveld A, Skavdis G, Mamalaki C, Grosveld F, Savakis C. 2001. In vivo transposition of Minos, a Drosophila mobile element, in mamma-

lian tissues. Proc Natl Acad Sci U S A 98:11474 –11478. Zambrowicz BP, Friedrich GA, Buxton EC, Lilleberg SL, Person C, Sands AT. 1998. Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells. Nature 392:608 –611. Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW Jr, Kipp P, Kohlhauff B, Ma ZQ, Markesich D,

Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C, Sands AT. 2003. Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc Natl Acad Sci U S A 100: 14109 –14114. Zhu H, Wang G, Li G, Han M, Xu T, Zhuang Y, Wu X. 2005. Ubiquitous expression of mRFP1 in transgenic mice. Genesis 42:86 –90.