Mol. Hum. Reprod. Advance Access originally published online on September 23, 2007
Molecular Human Reproduction 2007 13(11):771-779; doi:10.1093/molehr/gam069
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Gene trap mutagenesis: a functional genomics approach towards reproductive research
Division of Reproductive Biology Research, Department of Obstetrics and Gynecology, Center for Genetic Medicine, Feinberg School of Medicine, Northwestern University, Lurie 7-117, 303 E Superior Street, Chicago, IL 60611, USA
* Correspondence address. Tel: +1-312-503-0481; E-mail: e-xu{at}northwestern.edu
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Abstract |
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We have entered a new era of genomics in biomedical research with the availability of genome-wide sequences and expression data, resulting in the identification of a huge number of novel reproductive genes. The challenge we are facing today is how to determine the function of those novel and known genes and their roles in normal reproductive physiology, such as gamete production, pregnancy and fertilization, and the disease physiology such as infertility, spontaneous abortion and gynecological cancers. Mouse genetics has contributed tremendously to our understanding of the genetic causes of human diseases in the past decades. The establishment of mouse mutations is an effective way to understand the function of many reproductive proteins. One of the fast-growing mouse mutagenesis technologies—gene trap mutagenesis—represents a cost-effective way to generate mutations because of the public availability of mouse embryonic stem (ES) cell lines carrying insertional mutations and the continuing expansion of those ES gene trap cell lines. We review here the gene trapping technology and in particular examine its efficacy in generating mouse mutations for reproductive research. Even with the existing gene trap cell lines, many of the genes important for reproductive function through traditional knockout and chemical mutagenesis have been trapped, demonstrating gene trapping’s efficacy in mutating genes involved in reproductive development. Comparing genes expressed in specific reproductive sub-cellular organelles and in the entire testis and ovary with gene trap lines in the International Gene Trap Consortium (IGTC) database, we could identify a significant portion of those genes as having been trapped, representing a great resource for establishing mouse models for reproductive research. Establishment and analysis of these mouse models, for example, could help with identifying genetic abnormalities underlying male infertility and other reproductive diseases.
Key words: gene trap/mutation/male reproduction/spermatogenesis/acrosome
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Introduction |
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With the completion of the mapping of the human genome, we now have access to all the DNA sequence information responsible for human biology. Together with microarray technology, we are ushering in a new era in reproductive medicine—the era of reproductive genomics. Whole genome microarray analysis of the testis and ovary suggests that a substantial part of the genome is expressed in reproductive tissues and many of them are likely to be important for normal reproduction (Shima et al. 2004; Assou et al., 2006; Kocabas et al., 2006). Yet adequate expression and functional information is only available for less than 10% of them. Hence, one of the important questions in reproductive studies now is ‘how do we associate function with the genes expressed in reproductive tissues?’ The establishment of mutations in animal models such as the mouse represents one powerful approach to address this question.
Animal models have played critical roles in improving our understanding of mechanisms and pathogenesis of diseases. Mouse knockout models have often provided highly needed functional validation of genes implicated in human diseases. The rapid advance of human genetics in areas such as single nucleotide polymorphisms (SNP) and haplotyping technology now allows the identification of disease-associated single nucleotide variation at a much faster pace. Functional examination of those candidate genes is needed to determine if those genes or variants are indeed involved in reproductive disease. Generating mutations in murine homologs of candidate genes represents a direct way to determine their roles, and mouse models will further allow the dissection of genetic pathways underlying the disease condition and provide models to test possible drug treatments. Thus, how to generate mouse models efficiently becomes a priority issue in the genomics era of reproductive medicine.
It is known that generating a mouse knockout is no small endeavor, even for a mouse research lab, often requiring specialized expertise and experience in molecular biology, embryonic stem (ES) biology and mouse husbandry. Therefore, it could be intimidating for people who have little experience in mouse research. Fortunately, there are some technological developments in the mouse community that make the task of generating mouse mutations less intimidating to people unfamiliar with mouse genetics. One of these developments is the effort led by the International Gene Trap Consortium (IGTC) to generate a library of mouse mutant ES cells covering most of the genes in the mouse genome (Nord et al., 2006). This method saves researchers the tedious and sometimes challenging tasks of making knockout vectors and screening ES cell colonies and directly provides researchers an ES cell clone carrying the mutation of the gene of interest.
Because gene trapping involves the use of different mechanisms in generating mutations from the traditional knockout method, and its efficacy in targeting reproductive genes which often are expressed in later development or adults has not been fully established, it is necessary to examine the benefits and limitations of this technology, especially in the perspective of reproductive medicine so that reproductive researchers and physicians who are interested in mouse models could become familiar with this technology. With this in mind, we provide an overview of the gene trapping mutagenesis method and its possible application to reproductive medicine. We evaluate gene trapping as a method in terms of its efficiency in comparison with traditional knockout methods and use an in-house software program to screen the IGTC database for existing cell lines with possible mutations in genes expressed in various reproductive tissues. Among over seven thousand genes highly expressed in human ovaries, almost half of them have existing gene trap lines. Additionally, from 900 human seminal fluid proteins, 43% of them have gene trap hits in their mouse homologs. Our analysis suggests gene trapping is an effective mutagenesis method for identifying the genetic basis of reproductive diseases and many mutations for important reproductive genes are already present in the database. Given the rapid growth of the number of gene trap lines, the continuing evolution of gene trap vectors, and its easy accessibility to scientific communities, gene trapping could provide a fast and efficient way of generating mouse mutation(s) for any one particular gene of interest or multiple genes involved in a pathway at the same time. Consequently, we recommend gene trapping to be considered in the planning of mouse modeling of human reproductive disease and the IGTC be the first stop for people interested in searching for and generating mouse mutations of genes of interest.
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Gene Trapping: The Process |
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Gene trapping is a high-throughput approach of generating mutations in murine ES cells through vectors that simultaneously disrupt and report the expression of the endogenous gene at the point of insertion. First-generation vectors trapped genes that were actively transcribed in undifferentiated ES cells. Depending on the areas in which they integrate, these vectors can be roughly divided into two classes: promoter trap vectors and gene trap vectors. Promoter trap vectors contain promoterless reporter regions, usually ßgeo (a fusion of neomycin phosphotransferase and ß-galactosidase), and thus have to be integrated into an exon of a transcriptionally active locus in order for the cell to be selected for neomycin resistance or by LacZ staining (Skarnes et al., 1992; Friedrich and Soriano, 1993). Gene trap vectors demonstrate more utility by their added ability to integrate into an intron. These vectors contain a splice acceptor (SA) site positioned at the 5'-end of the reporter gene, allowing the vector to be spliced to the endogenous gene to form a fusion transcript (Fig. 1). Later improvements include an internal ribosomal re-entry site (IRES) between the SA site and the reporter gene sequence; as a result, the reporter gene can be translated even when it is not fused to the trapped gene (Lako and Hole, 2000).
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Second-generation vectors have sought to trap genes that are transcriptionally silent in ES cells. Although these vectors still contain a promoterless reporter gene with a 5' SA sequence, the antibiotic resistance gene is under the control of a constitutive promoter. Consequently, antibiotic selection is independent from the expression of the trapped gene, whereas the expression of the reporter gene is still regulated by the endogenous promoter (Gossler et al., 1989; Niwa et al., 1993; Salminen et al., 1998; Forrai and Robb, 2005). A disadvantage of these vectors is that all integration events give rise to resistant ES cells regardless of whether or not the vector has integrated into a gene locus. To increase trapping efficiency, a new class of polyA gene trap vectors was developed where the polyadenylation signal of the neo gene was replaced by a splice donor sequence, thereby requiring the vector to trap an endogenous polyA signal for expression of neo (Niwa et al., 1993; Salminen et al., 1998). These vectors were recently shown to have a bias toward insertion near the 3'-end of a gene due to nonsense-mediated mRNA decay of the fusion transcript. An improved polyA trap vector, UPATrap, was developed to overcome this bias using an IRES sequence placed downstream of a marker containing a termination codon (Shigeoka et al., 2005).
Gene trap vectors are usually introduced by retroviral infection or electroporation of plasmid DNA, with each approach having its own advantages and disadvantages. While relatively difficult to manipulate, retroviral gene traps display a preference toward insertion at the 5'-end of genes, which is advantageous for generating null alleles (Wu et al., 2003; De-Zolt et al., 2006). Moreover, the multiplicity of infection with retroviruses can be tightly controlled to a single trap event or simultaneous disruption in many genes. However, there may be a possible bias integration toward certain ‘hotspots’ of the genome (Hansen et al., 2003; Chen et al., 2004; Raymond and Soriano, 2006). In contrast, plasmid-based gene trap vectors integrate more randomly into the genome. This can, however, potentially result in a functional partial protein and a hypomorphic phenotype (Raymond and Soriano, 2006). Additionally, plasmid vectors usually result in multiple integrations in 20–50% of cell lines (Wiles et al., 2000; Forrai and Robb. 2005).
The most common approach for identifying the gene trap integration site is to use 5' or 3' rapid amplification of cDNA ends (RACE) to amplify the fusion transcript (Pires-DaSilva and Gruss, 1998; Yoshida et al., 1995; Wiles et al., 2000; Forrai and Robb, 2005). The sequence provides a DNA tag for the identification of the disrupted gene and can be used for genotypic screens. Mutagenesis screens can also be performed on the basis of gene function or expression, and data from an expression sequence combined with sequence tag information can elucidate novel expression patterns of known genes or to suggest gene function (Skarnes et al., 1992; Bonaldo et al., 1998; Hirashima et al., 2004; Forrai and Robb, 2005; Shirasawa et al., 2005).
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IGTC: An Online Resource for Gene Trap Data |
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Various laboratories worldwide have conducted large-scale efforts to generate gene trap ES cell lines, with the ultimate goal of trapping every gene in the murine genome. In an effort to consolidate this data, the IGTC has been created with the goal of providing a free library of all publicly-available gene trap cell lines from its members: BayGenomics, Centre for Modelling Human Disease, Embryonic Stem Cell Database, Exchangeable Gene Trap Clones, German Gene Trap Consortium, Sanger Institute Gene Trap Resource, Soriano Lab Gene Trap Database and TIGEM-IRBM Gene Trap. To date, the IGTC has generated more than 57 000 cell lines, representing over 9000 known mouse mutations or
40% of known mouse genes (Raymond and Soriano, 2006). The sequence tags for the trapped cell lines are fully annotated in a user-friendly, searchable website located at the IGTC website (http://www.genetrap.org), which also contains features such as useful documentation, the ability to view trapped genes within biological pathways and the ability to search by expression profile (Nord et al., 2006). In addition, the sequence tags are mapped on the ensemble mouse genome browser (http://www.ensembl.org/Mus_musculus) under the distributed annotation system (DAS) source ‘Gene Trap’ (Skarnes et al., 2004). The development of the IGTC website marks a significant advance by providing the research community with the data and tools necessary to utilize public gene trap resources for the large-scale characterization of mammalian gene function. The ability to efficiently trap, sequence and detect the expression of genes, regardless of their transcriptional activity, has made gene trapping an exceptional tool for gene discovery, especially with the establishment of the IGTC. Moreover, the versatility of this approach has been improved by the development of vectors that include recombination or promoter sites (Araki et al., 1999; Hardouin and Nagy, 2000). Therefore, gene trapping has been utilized in many areas of research, including apoptosis, neurology and hematopoiesis (Wempe et al., 2001; Yin et al., 2003; Forrai and Robb, 2005). Reproductive research also has great potential to benefit from this method of mutagenesis, and it is within this specific context that we evaluated the efficacy of gene trapping with the goal of using this resource to perform functional analyses of genes involved with reproduction.
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SpiderGene: A High-Throughput Database Webspider |
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 TopAbstractIntroductionGene Trapping: The ProcessIGTC: An Online Resource... SpiderGene: A High-Throughput... Application to Reproductive...ConclusionSupplementary DataFundingAcknowledgementsReferences |
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Investigators in the reproductive field seeking to use gene trapping encounter several difficulties. Gene symbols are somewhat variable and referencing them is an unreliable way to search the IGTC database; a more favorable way is to do a BLAST search of corresponding DNA sequences. Moreover, a substantial amount of time and effort is required to obtain a FASTA-formatted list of DNA sequences, perform a BLAST search on the IGTC database and sort through the results to identify valid gene trap lines. Therefore, our group developed a program in Visual Basic named 'SpiderGene' (http://www.feinberg.northwestern.edu/xulab) in order to minimize or eliminate these limitations, greatly facilitating our investigation. It is designed to accept as user input a spreadsheet list of gene symbols, GeneBank Accession numbers or Unigene cluster numbers. The program will then proceed to search National Center for Biotechnology Information (NCBI)’s Unigene database, navigating through the Unigene cluster browser to the GenBank database and extracting the DNA sequence for the best expressed sequence tags (EST) sequence for each particular gene (Benson et al., 2006). This seemingly roundabout path also allows the user to use Unigene’s EST expression profile data to narrow down genes based on their expression profile if desired.
The sequences are then placed in FASTA format, which the user can use to BLAST the IGTC database. After results are generated, the user can paste the entire page of results into a window in SpiderGene, which will proceed to sort through the results based on a user-defined criterion of minimum bit score denoting a match. The end result is a list of genes fitting the criteria for a valid gene trap hit. Although the advantages of the use of SpiderGene are primarily to save time and to decrease the amount of false negatives on the basis of using DNA sequences, there may be a number of false positives due to sequence similarity of genes of the same gene family or genes sharing a highly conserved protein motif. The number of false positives may become substantial when gene symbols are used as a result of unavoidable idiosyncrasies of NCBI’s search function. However, accuracy greatly increases with the use of GenBank accession numbers or Unigene sequence cluster numbers. It should be noted that for most major centers, the sequencing and annotation process of trapped genes is an automated process. Therefore, recognizing the possibility of false positives, IGTC recommends that the researcher download the sequence tag and individually confirm the hit for correct annotation. Once the hits are confirmed, the user can follow the links provided on the IGTC site to order the cell lines from the research group that developed the line.
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Application to Reproductive Research |
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Gene trapping has been shown to be effective in generating mutations affecting developmental processes in early embryonic development (Leighton et al., 2001; Mitchell et al., 2001; Reiter and Skarnes, 2006), but its application in reproductive research remains largely unexplored. There are several reasons that might contribute to the hesitation among reproductive researchers and clinicians to embrace this new technology.
- (i) The concern of gene trapping bias against developmental processes in later development such as reproductive processes. Because mouse ES gene trapping technology has been relying on mouse ES cells for screening gene trapping mutation, the genes of interest have to be expressed in the mouse ES cells in order for them to be trapped in the screening. It is not surprising that many embryonic developmental regulators are efficiently mutated with this method, but its efficiency in trapping genes involved in adult stages is not clear. This concern of using ES-based gene trap method for knockout becomes more prominent when one considers that many reproductive processes are taking place later during development and even restricted in the adult stage.
- (ii) Concerns of differences in splicing between reproductive tissues and embryonic tissues. Unlike traditional gene targeting which removes a part of a gene from genomic DNA, the mutation of gene trapping does not become effective until the inserted vector is spliced together with the exons next to where the vector is inserted. It is well known that many genes have testis or ovary specific splicing isoforms in addition to the common forms. It is difficult to predict if a gene trap vector, which is spliced into the gene of interest in ES cells to cause mutation, will disrupt the testis or ovary isoforms.
- (iii) Concerns over genes specifically expressed in the reproductive tissues. Often genes specifically expressed in the reproductive tissues are of much interest. It also raises questions of whether genes with such restricted expression pattern could still be trapped in mouse ES cells.
- (iv) How useful is this approach to reproductive research in general? All the mutagenic methods have limitations and so does the gene trapping method. Are there any types of genes not mutatable by this method and how much coverage of reproductive genes is in the current gene trap ES cell database?
- (ii) Concerns of differences in splicing between reproductive tissues and embryonic tissues. Unlike traditional gene targeting which removes a part of a gene from genomic DNA, the mutation of gene trapping does not become effective until the inserted vector is spliced together with the exons next to where the vector is inserted. It is well known that many genes have testis or ovary specific splicing isoforms in addition to the common forms. It is difficult to predict if a gene trap vector, which is spliced into the gene of interest in ES cells to cause mutation, will disrupt the testis or ovary isoforms.
We have hence set out to examine this gene trapping method and the pros and cons in its potential application to reproductive research.
How efficient is gene trapping compared to traditional knockout methods?
To compare the efficacy of gene trapping against other mutagenesis strategies in reproductive research, we used a list of 202 reported mouse reproductive mutations (Matzuk and Lamb, 2002) and asked how many of those genes with reproductive function have been targeted in the current gene trap ES cell database. These mutations cause various reproductive defects and the mouse models were generated by various mutagenesis schemes such as spontaneous mutations, fortuitous transgene integration, retroviral infection of ES cells, ethylnitrosurea (ENU) mutagenesis and gene targeting technologies (Matzuk and Lamb, 2002). After processing these genes with SpiderGene, results revealed that 76 genes or 38% of the list have been trapped using gene trap mutagenesis. Table 1 highlights some of the well-known genes (for a complete list of genes trapped, see the Supplementary Table). This suggests that genes involved in reproductive function could be targeted using gene trapping mutagenesis. Gene trapping technology alone has targeted more than one-third of known reproductive genes, which have been mutated through all kinds of mutagenesis methods in the past several decades. This is affirming evidence to conclude that gene trapping is a valid way to approach functional analysis in reproductive research, and this percentage will continue to increase with the new generation of vectors and improved efficiency as well as rapidly expanding cell lines.
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What percentage of genes involved in reproduction is trapped?
The promise of gene trapping mutagenesis is not only that it is a potent mutagenic method but also that it could provide a way for functional studies on a large scale, or a functional genomics approach to mammalian biology. We hence utilize gene expression data for different reproductive tissues to search for gene trap hits in the IGTC database and ask if we could use gene trap ES cell lines in performing a functional genomics study in reproductive medicine.
We start first with a subcellular compartment of sperm, the acrosome, and ask how many of the acrosomal components have been trapped. The acrosome is a secretory organelle required for normal fertilization. Identifying the function of acrosomal components could reveal insights into sperm–oocyte interaction and provide novel targets for male contraceptive development. Stein et al. (2006) has used the fractionation method to purify proteins from the sperm acrosome and to perform proteomics analysis on these proteins. They reported a list of 54 proteins as contents of the acrosome including enzymes and enzyme inhibitors. Among them, the functions of only nine of them are known from mouse models. We hence ran all 54 proteins in our software and found 16 ES gene trap lines for those acrosomal components (30%). Together with the traditional method, we could achieve 37% coverage of the reported acrosomal proteins. We then use another key part of sperm, the flagellum, as our target sperm compartment to see how many proteins in the sperm flagellum have been trapped. Our results found that among 26 proteins reported by proteomic analysis of mouse sperm, gene trap lines exist for five of them (Cao et al., 2006). Thus, even considering only two subcellular components of mature spermatozoa, ES gene trap mutagenesis effectively mutates 21 protein components of sperm, representing 26% coverage.
Another important component of the fertilization process is seminal fluid, which carries sperm during its travel to meet the oocyte. Among 900 seminal fluid proteins reported, we searched the IGTC database and identified 386 gene trap lines at a rate of 43% (Pilch and Mann, 2006). This suggests that almost half of the seminal fluid proteins are trapped and those gene trap lines represent excellent resources for the functional analysis of seminal fluid proteins in fertility.
We further examined a recent report of the presence of RNA in mature spermatozoa and found that only 31 of the genes are trapped (4%). The relatively low gene trap efficiency suggests that those spermatozoa transcripts are much less likely to be expressed in ES cells and hence have a lower percentage of trapping rate.
Sperm production is a complex process involving many different cell types and going through different stages of cells development. Genes involved in different stages of development now can be identified using microarrays. We chose one specific stage—human premeiotic germ cell development—and the genes highly expressed in the entire mouse testis to examine how the gene trapping scheme targets developmentally regulated genes during spermatogenesis. Among 65 X-linked genes known to be expressed in premeiotic germ cell development (Koslowski et al. 2006), 11 or about 17% have positive hits in the IGTC database. The next question we asked is how many of genes expressed in the entire testis could be trapped. Since whole genome analysis on mouse testis has been reported and over 11 thousand genes are shown to be differentially expressed during different stages of spermatogenesis (Shima et al., 2004), we searched the entire IGTC database for possible hits. We identified 2920 hits in the IGTC database, indicating one-quarter of genes expressed in the testis have been trapped. The almost three thousand gene trap ES cell lines carrying insertions in those potentially important spermatogenic genes represent an excellent resource for fertility and male contraception research.
Although we have focused on the male reproductive tissues with our analysis, ES gene trapping is equally effective when it comes to female reproductive tissues. We use the list of more than three thousand genes specifically expressed in the human ovaries and we found that more than half of the genes have been trapped (Kocabas et al., 2006) (1797 out of 3267, Fig. 2), suggesting that the gene trap method is effective in trapping genes expressed in both male and female reproductive tissues. This large number of gene trap ES cell lines containing mutations of genes important for gamete function and the normal development of male and female gametes provides an excellent resource for functional genomics analysis in our reproductive research. The study of these genes has a substantial implication in our research on the development of contraceptives, genetic analysis of fertility and other important reproductive issues.
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Which trapped genes are specifically expressed in the reproductive tissue?
One of the interesting areas to reproductive biologists is the functional analysis of reproductive specific genes. Although we know from the above analysis that many reproductive genes have been trapped, including genes with known reproductive function, it is not known whether genes with restricted expression in testis or ovary could be trapped and whether those trapped reproductive genes are genes with expression in many different tissues including ES cells. One specific goal for our laboratory was to identify potential candidates that play a crucial and specific role in germ line development, as we hope to reveal potential targets for contraceptive drug development. Therefore, we added an additional feature to SpiderGene that parses through the expression profile data available from Unigene to narrow down candidate trapped cell lines on the basis of EST numbers for genes possessing localized or predominant expression in the reproductive tissue (testis and ovary). These results are included in Tables 2 and 3. We are clearly able to identify gene trap hits in genes, which are only expressed or predominantly expressed in the testis through this method. The percentage of hits is relatively low, reflecting that those reproductive specific genes are being trapped but at a lower frequency. With the constant increasing number of gene trap ES cell lines deposited everyday, we should expect more ES cell lines from this category.
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Mouse mutations generated from gene trap lines inserted into known reproductive genes
Several groups have already used the gene trap resource in the field of reproductive research. Examples of these genes are reported in Table 4. Analysis of genes such as these, aided by mouse models established from gene trap cell lines, can provide valuable information regarding the underlying genetic mechanism and etiology of reproductive abnormalities such as non-obstructive azoospermia. Additionally, Kanatsu-Shinohara et al. (2006) have recently reported successful gene trapping and homologous recombination in spermatogonial stem cells, not only creating another avenue for reproductive research but also demonstrating the feasibility of altering genes in a tissue-specific manner (Kanatsu-Shinohara et al., 2006).
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Advantages and limitations of gene trap mutagenesis
Gene trapping is an exceptional tool for gene discovery and our bio-informatic analyses further show that gene trap mutagenesis represents a powerful tool for reproductive genetics and for reproductive disease modeling. However, we need to evaluate gene trap in the context of other mouse functional genomics techniques. The advantages of gene trapping are that it is gene-oriented and the mutated gene is known from the beginning due to its reverse genetics approach. The reporter gene could also be used to reveal the expression pattern of the gene of interest. This is particularly useful for those genes that are developmentally regulated and expressed in different cell types and at different stages of reproductive development. Another unique advantage is the ease of gene trap to establish an allelic series consisting of hypomorphic, loss-of-function allele and even gain-of-function allele with the different gene trap lines for the same gene. This is especially powerful for mutating genes essential for embryonic development and the reproductive process, as hypomorphic allele may not impair embryonic development and could allow the mutants to develop to the adult stage when it is possible to examine the reproductive defects of the mutations.
There are several limitations with gene trapping one should be aware of before starting experiments with gene trapping ES cell lines. First the phenotypes of the mutants are unpredictable. Although the expression of a particular gene in a reproductive tissue of interest may be a clue for a possible function, it may not always be the case. This is similar to other reverse genetics mutagenesis methods such as gene targeting. The derived mutant mice may not exhibit any reproductive defects if its function is not essential for reproduction. The more biology is known about the gene, the better it will be to predict the phenotype of the loss-of-function mutation. Second, common gene trap methods rely on the expression of genes in ES cells, and the genes which are not expressed in ES cells hence cannot be trapped by this method. However, the majority of genes in the genome are expressed in undifferentiated ES cells, which includes many reproductive genes as illustrated in this article. It is estimated that 60% of genes from the mouse genome have been trapped and it has become increasingly difficult to trap the remaining 40% due to their low expression levels in undifferentiated ES cells with the standard gene trapping method alone (Skarnes, 2005; Skarnes et al., 2004). The continuing evolving technology and use of a variety of plasmids and vectors could help to trap the remaining genes in the genome. For example, a recent system capable of reporting conditional expression of a gene in a tissue- or temporal-specific pattern has been developed using transposable elements, particularly the Sleeping Beauty (SB) transposon (Carlson et al., 2003). Several groups have reported the success of in vivo functionality of the SB vector and its utility in insertional mutagenesis (Carlson et al., 2003; Clark et al., 2004; Geurts et al., 2006).
Third, one major shortcoming of gene trap approach is that it may not always produce a loss-of-function mutation as alternative splicing could happen, resulting in low levels of wild-type transcripts and often a hypomorphic allele. One way to minimize such risk is to work with two or more different ES cell lines with insertions in different parts of the gene at the same time in order to increase the chance of obtaining a complete loss-of-function allele. Recently, a targeted gene trapping approach is reported to efficiently inactivate genes expressed in ES cells with an average targeting efficiency of above 50% (Friedel et al., 2005; Skarnes, 2005). The simplicity and high efficiency of this targeted gene trapping method could facilitate investigators in generating a loss-of-function allele of their targeted genes.
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Conclusion |
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Gene trapping has proven to be an efficacious technique in mutagenesis compared with other methods such as spontaneous mutations, fortuitous transgene integration and N-ethyl-N-nitrosurea (ENU) mutagenesis. We have been able to use our SpiderGene program to identify genes in reproductive tissues that are present in the IGTC database and moreover to narrow down those with restricted expression in the testis and ovary. Gene trapping possesses an enormous potential for researchers in the reproductive field seeking to create mouse models for a gene mutation. The improving versatility of gene trap vectors has enabled groups to trap an increasing number of genes in various organisms, including Arabidopsis, Zebra fish and Drosophila (Lukacsovich and Yamamoto. 2001; Kotani et al., 2006; Nagawa et al., 2006). The gene trap effort has perhaps been the most extensive in the murine genome, with over 57 000 cell lines representing more than 40% of the known genome (Raymond and Soriano, 2006). These large-scale screens will likely achieve the trapping of the entire mouse genome in the coming years, but the power of gene trapping will only be fully demonstrated by its usefulness in investigator-driven focused functional analyses. In our laboratory, future work will focus on generating knockout mice in order to investigate gene function and to identify gene products that might have therapeutic value in reproduction. As screening efforts continue, gene trapping will continue to be a valuable tool in mouse genomics and will undoubtedly yield new discoveries in reproductive physiology and pathology.
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Supplementary Data |
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Supplementary data are available at http://molehr.oxfordjournals.org/.
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Funding |
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NIH (U01 HD045871) and by EAM grant from the State of Illinois, Department of Public Aid and Northwestern Memorial Hospital.
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Acknowledgements |
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The authors are grateful to Yanmei Chen, Villian Naeem and Yin Wang for assistance in the laboratory and Drs Erwin Goldberg, Nicholas Salmon and Robert Brannigan for comments on the manuscript.
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References |
|---|
Araki K, Imaizumi T, Sekimoto T, Yoshinobu K, Yoshimuta J, Akizuki M, Miura K, Araki M, Yamamura K. Exchangeable gene trap using the Cre/mutated lox system. Cell Mol Biol (Noisy-le-grand) (1999) 45:737–750.[Medline]
Assou S, Anahory T, Pantesco V, Le Carrour T, Pellestor F, Klein B, Reyftmann L, Dechaud H, De Vos J, Hamamah S. The human cumulus–oocyte complex gene-expression profile. Hum Reprod (2006) 21:1705–1719.
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL. GenBank. Nucleic Acids Res (2006) 34:D16–D20.
Bergo MO, Gavino BJ, Steenbergen R, Sturbois B, Parlow AF, Sanan DA, Skarnes WC, Vance JE, Young SG. Defining the importance of phosphatidylserine synthase 2 in mice. J Biol Chem (2002) 277:47701–47708.
Bonaldo P, Chowdhury K, Stoykova A, Torres M, Gruss P. Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection. Exp Cell Res (1998) 244:125–136.[CrossRef][Web of Science][Medline]
Campbell PK, Waymire KG, Heier RL, Sharer C, Day DE, Reimann H, Jaje JM, Friedrich GA, Burmeister M, Bartness TJ, et al. Mutation of a novel gene results in abnormal development of spermatid flagella, loss of intermale aggression and reduced body fat in mice. Genetics (2002) 162:307–320.
Cao W, Gerton GL, Moss SB. Proteomic profiling of accessory structures from the mouse sperm flagellum. Mol Cell Proteomics (2006) 5:801–810.
Carlson CM, Dupuy AJ, Fritz S, Roberg-Perez KJ, Fletcher CF, Largaespada DA. Transposon mutagenesis of the mouse germline. Genetics (2003) 165:243–256.
Chen WV, Delrow J, Corrin PD, Frazier JP, Soriano P. Identification and validation of PDGF transcriptional targets by microarray-coupled gene-trap mutagenesis. Nat Genet (2004) 36:304–312.[CrossRef][Web of Science][Medline]
Chennathukuzhi V, Stein JM, Abel T, Donlon S, Yang S, Miller JP, Allman DM, Simmons RA, Hecht NB. Mice deficient for testis-brain RNA-binding protein exhibit a coordinate loss of TRAX, reduced fertility, altered gene expression in the brain, and behavioral changes. Mol Cell Biol (2003) 23:6419–6434.
Clark KJ, Geurts AM, Bell JB, Hackett PB. Transposon vectors for gene-trap insertional mutagenesis in vertebrates. Genesis (2004) 39:225–233.[CrossRef][Web of Science][Medline]
Couldrey C, Carlton MB, Nolan PM, Colledge WH, Evans MJ. A retroviral gene trap insertion into the histone 3.3A gene causes partial neonatal lethality, stunted growth, neuromuscular deficits and male sub-fertility in transgenic mice. Hum Mol Genet (1999) 8:2489–2495.
De-Zolt S, Schnutgen F, Seisenberger C, Hansen J, Hollatz M, Floss T, Ruiz P, Wurst W, von Melchner H. High-throughput trapping of secretory pathway genes in mouse embryonic stem cells. Nucleic Acids Res (2006) 34:e25.
Forrai A, Robb L. The gene trap resource: a treasure trove for hemopoiesis research. Exp Hematol (2005) 33:845–856.[CrossRef][Web of Science][Medline]
Friedel RH, Plump A, Lu X, Spilker K, Jolicoeur C, Wong K, Venkatesh TR, Yaron A, Hynes M, Chen B, et al. 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 USA (2005) 102:13188–13193.
Friedrich G, Soriano P. Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells. Methods Enzymol (1993) 225:681–701.[Web of Science][Medline]
Geurts AM, Wilber A, Carlson CM, Lobitz PD, Clark KJ, Hackett PB, McIvor RS, Largaespada DA. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol (2006) 6:30.[CrossRef][Medline]
Gossler A, Joyner AL, Rossant J, Skarnes WC. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science (1989) 244:463–465.
Hansen J, Floss T, Van Sloun P, Fuchtbauer EM, Vauti F, Arnold HH, Schnutgen F, Wurst W, von Melchner H, Ruiz P. A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. Proc Natl Acad Sci USA (2003) 100:9918–9922.
Hardouin N, Nagy A. Gene-trap-based target site for cre-mediated transgenic insertion. Genesis (2000) 26:245–252.[CrossRef][Web of Science][Medline]
Hirashima M, Bernstein A, Stanford WL, Rossant J. Gene-trap expression screening to identify endothelial-specific genes. Blood (2004) 104:711–718.
Hosaka T, Biggs WH 3rd, Tieu D, Boyer AD, Varki NM, Cavenee WK, Arden KC. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc Natl Acad Sci USA (2004) 101:2975–2980.
Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, Inoue K, Kazuki Y, Lee J, Toyokuni S, Oshimura M, et al. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc Natl Acad Sci USA (2006) 103:8018–8023.
Kocabas AM, Crosby J, Ross PJ, Otu HH, Beyhan Z, Can H, Tam WL, Rosa GJ, Halgren RG, Lim B, et al. The transcriptome of human oocytes. Proc Natl Acad Sci USA (2006) 103:14027–14032.
Komada M, McLean DJ, Griswold MD, Russell LD, Soriano P. E-MAP-115, encoding a microtubule-associated protein, is a retinoic acid-inducible gene required for spermatogenesis. Genes Dev (2000) 14:1332–1342.
Koslowski M, Sahin U, Huber C, Tureci O. The human X chromosome is enriched for germline genes expressed in premeiotic germ cells of both sexes. Hum Mol Genet (2006) 15:2392–2399.
Kotani T, Nagayoshi S, Urasaki A, Kawakami K. Transposon-mediated gene trapping in zebrafish. Methods (2006) 39:199–206.[CrossRef][Web of Science][Medline]
Lako M, Hole N. Searching the unknown with gene trapping. Expert Rev Mol Med (2000) 2000:1–11.[Medline]
Leighton PA, Mitchell KJ, Goodrich LV, Lu X, Pinson K, Scherz P, Skarnes WC, Tessier-Lavigne M. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature (2001) 410:174–179.[CrossRef][Medline]
Lukacsovich T, Yamamoto D. Trap a gene and find out its function: toward functional genomics in Drosophila. J Neurogenet (2001) 15:147–168.[Web of Science][Medline]
Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways. Nat Cell Biol (2002) 4(Suppl.):s41–s49.[Web of Science][Medline]
Mitchell KJ, Pinson KI, Kelly OG, Brennan J, Zupicich J, Scherz P, Leighton PA, Goodrich LV, Lu X, Avery BJ, et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat Genet (2001) 28:241–249.[CrossRef][Web of Science][Medline]
Nagawa S, Sawa S, Sato S, Kato T, Tabata S, Fukuda H. Gene trapping in Arabidopsis reveals genes involved in vascular development. Plant Cell Physiol (2006) 47:1394–1405.
Nayernia K, Vauti F, Meinhardt A, Cadenas C, Schweyer S, Meyer BI, Schwandt I, Chowdhury K, Engel W, Arnold HH. Inactivation of a testis-specific Lis1 transcript in mice prevents spermatid differentiation and causes male infertility. J Biol Chem (2003) 278:48377–48385.
Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K. An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events. J Biochem (Tokyo) (1993) 113:343–349.
Nord AS, Chang PJ, Conklin BR, Cox AV, Harper CA, Hicks GG, Huang CC, Johns SJ, Kawamoto M, Liu S, et al. The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse. Nucleic Acids Res (2006) 34:D642–D648.
Osada T, Watanabe G, Kondo S, Toyoda M, Sakaki Y, Takeuchi T. Male reproductive defects caused by puromycin-sensitive aminopeptidase deficiency in mice. Mol Endocrinol (2001) 15:960–971.
Ostermeier GC, Dix DJ, Miller D, Khatri P, Krawetz SA. Spermatozoal RNA profiles of normal fertile men. Lancet (2002) 360:772–777.[CrossRef][Web of Science][Medline]
Pilch B, Mann M. Large-scale and high-confidence proteomic analysis of human seminal plasma. Genome Biol (2006) 7:R40.[CrossRef][Medline]
Pinson KI, Brennan J, Monkley S, Avery BJ, Skarnes WC. An LDL-receptor-related protein mediates Wnt signalling in mice. Nature (2000) 407:535–538.[CrossRef][Medline]
Pires-DaSilva A, Gruss P. Gene trap insertion into a novel gene expressed during mouse limb development. Dev Dyn (1998) 212:318–325.[CrossRef][Web of Science][Medline]
Pires-daSilva A, Nayernia K, Engel W, Torres M, Stoykova A, Chowdhury K, Gruss P. Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities. Dev Biol (2001) 233:319–328.[CrossRef][Web of Science][Medline]
Raymond CS, Soriano P. Engineering mutations: Deconstructing the mouse gene by gene. Dev Dyn (2006) 235:2424–2436.[CrossRef][Web of Science][Medline]
Reiter JF, Skarnes WC. Tectonic, a novel regulator of the Hedgehog pathway required for both activation and inhibition. Genes Dev (2006) 20:22–27.
Ross AJ, Waymire KG, Moss JE, Parlow AF, Skinner MK, Russell LD, MacGregor GR. Testicular degeneration in Bclw-deficient mice. Nat Genet (1998) 18:251–256.[CrossRef][Web of Science][Medline]
Salminen M, Meyer BI, Gruss P. Efficient poly A trap approach allows the capture of genes specifically active in differentiated embryonic stem cells and in mouse embryos. Dev Dyn (1998) 212:326–333.[CrossRef][Web of Science][Medline]
Salmon NA, Reijo Pera RA, Xu EY. A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality. Genesis (2006) 44:515–522.[CrossRef][Web of Science][Medline]
Shigeoka T, Kawaichi M, Ishida Y. Suppression of nonsense-mediated mRNA decay permits unbiased gene trapping in mouse embryonic stem cells. Nucleic Acids Res (2005) 33:e20.
Shima JE, McLean DJ, McCarrey JR, Griswold MD. The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biol Reprod (2004) 71:319–330.
Shirasawa S, Yoshimi M, Kamochi H, Muneta T, Kimura JH, Hook M, Shinomura T. Gene trap screening for cell surface and extracellular matrix molecules produced by chondrocytes. J Biochem (Tokyo) (2005) 137:79–85.
Skarnes WC. Two ways to trap a gene in mice. Proc Natl Acad Sci USA (2005) 102:13001–13002.
Skarnes WC, Auerbach BA, Joyner AL. 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 (1992) 6:903–918.
Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, et al. A public gene trap resource for mouse functional genomics. Nat Genet (2004) 36:543–544.[CrossRef][Web of Science][Medline]
Stein KK, Go JC, Lane WS, Primakoff P, Myles DG. Proteomic analysis of sperm regions that mediate sperm-egg interactions. Proteomics (2006) 6:3533–3543.[CrossRef][Web of Science][Medline]
Sutherland HG, Newton K, Brownstein DG, Holmes MC, Kress C, Semple CA, Bickmore WA. Disruption of Ledgf/Psip1 results in perinatal mortality and homeotic skeletal transformations. Mol Cell Biol (2006) 26:7201–7210.
Tanaka H, Iguchi N, Toyama Y, Kitamura K, Takahashi T, Kaseda K, Maekawa M, Nishimune Y. Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function. Mol Cell Biol (2004) 24:7958–7964.
Wempe F, Yang JY, Hammann J, von Melchner H. Gene trapping identifies transiently induced survival genes during programmed cell death. Genome Biol (2001) 2. RESEARCH0023.
Wiles MV, Vauti F, Otte J, Fuchtbauer EM, Ruiz P, Fuchtbauer A, Arnold HH, Lehrach H, Metz T, von Melchner H, et al. Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat Genet (2000) 24:13–14.[CrossRef][Web of Science][Medline]
Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science (2003) 300:1749–1751.
Xu EY, Chang R, Salmon NA, Reijo Pera RA. A gene trap mutation of a murine homolog of the Drosophila stem cell factor Pumilio results in smaller testes but does not affect litter size or fertility. Mol Reprod Dev (2007) 74:912–921.[CrossRef][Web of Science][Medline]
Yin Y, Kikkawa Y, Mudd JL, Skarnes WC, Sanes JR, Miner JH. Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques. Genesis (2003) 36:114–127.[CrossRef][Web of Science][Medline]
Yoshida M, Yagi T, Furuta Y, Takayanagi K, Kominami R, Takeda N, Tokunaga T, Chiba J, Ikawa Y, Aizawa S. A new strategy of gene trapping in ES cells using 3'RACE. Transgenic Res (1995) 4:277–287.[CrossRef][Web of Science][Medline]
Submitted on July 13, 2007; resubmitted on August 21, 2007; accepted on September 13, 2007.
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