Briefings in Functional Genomics and Proteomics Advance Access originally published online on February 22, 2006
Briefings in Functional Genomics and Proteomics 2006 5(1):15-18; doi:10.1093/bfgp/ell009
© Oxford University Press, 2006, All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
Generation of multipurpose alleles for the functional analysis of the mouse genome
Frank Schnütgen
Frank Schnütgen, Department of Molecular Hematology, University of Frankfurt Medical School, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany. Tel: +49 69 6301 4941; Fax: +49 69 6301 6390. E-mail: schnuetgen{at}em.uni-frankfurt.de
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ABSTRACT
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A novel generation of retroviral gene-trap vectors has been
developed with the ability to induce conditional mutations in
most genes expressed in mouse embryonic stem (ES) cells. The
vectors rely on directional site-specific recombination systems,
which can repair and re-induce the gene-trap mutations when
activated in succession. After the gene-trap insertions are
passaged into mice, this system enables the induction of temporally
and spatially restricted mutations in somatic cells. In addition
to their conditional features, the vectors create multipurpose
alleles amenable to a wide range of post-insertional modifications.
These vectors have been used to assemble the largest library
of ES cell lines with conditional mutations in single genes,
presently totalling 1724 unique genes. Due to their efficiency,
the vectors are part of the core technologies to be used by
EUCOMM for establishing a complete collection of conditional
null mutations in mice.
Keywords: gene trap, Cre recombinase, FLPe recombinase, recombination, conditional, cassette exchange
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INTRODUCTION
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With the completion of sequencing of the human and mouse genomes,
geneticists’ attention has shifted towards the functional
annotation of every single gene in the mammalian genome [
1,
2]. Among a multitude of approaches addressing gene function,
mutagenesis in the mouse is presently the most relevant for
extrapolation of human genetic disease. Compared with other
model organisms, the mouse offers particular advantages because
its genome structure and organization are closely related to
the human genome. Most importantly, the availability of mouse
embryonic stem (ES) cells, which grow indefinitely in tissue
culture, allows the generation of mice with defined mutations
in single genes for functional analysis and studies of human
disease.
Gene trapping is a high-throughput approach that is used to introduce insertional mutations across the mouse genome. It is performed with gene-trap vectors whose principal element is a gene-trapping cassette consisting of a promoterless reporter gene and/or selectable marker gene flanked by an upstream 3' splice site (splice acceptor; SA) and a downstream transcriptional termination sequence (polyadenylation sequence; polyA). When inserted into an intron of an expressed gene, the gene-trap cassette is transcribed from the endogenous promoter in the form of a fusion transcript in which the exon(s) upstream of the insertion site is spliced in frame to the reporter/selectable marker gene. Since transcription is terminated prematurely at the inserted polyadenylation site, the processed fusion transcript encodes a truncated and non-functional version of the cellular protein and the reporter/selectable marker [3]. Thus, gene traps simultaneously inactivate and report the expression of the trapped genes at the insertion sites, and provide DNA tags (gene-trap sequence tag; GTST) for the rapid identification of the disrupted genes. As gene-trap vectors insert randomly across the genome, a large number of mutations can be induced in ES cells within a limited number of experiments. Gene-trap approaches have been successfully used in the past by both academic and private organizations to create libraries of ES cell lines harbouring mutations in single genes [4–7]. Collectively, the existing resources cover about 66% of all protein-coding genes within the mouse genome [8].
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CONDITIONAL GENE TRAPPING
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Most gene-trap vectors, employed for generating currently available
resources, induce only ‘null’ mutations so that
mouse mutants generated from these libraries can report only
the earliest and non-redundant developmental function of the
trapped gene. Consequently, for most of the mutant strains generated
from these cell lines the significance of the trapped gene for
human disease remains uncertain, as most human disorders result
from late onset gene dysfunction. In addition, between 20 and
30% of the genes targeted in ES cells are required for development
and cause embryonic lethal phenotypes when passaged to the germline,
which precludes their functional analysis in the adult [
5,
9].
To address this problem, we have developed a novel generation of gene-trap vectors [10] with the ability to induce conditional knockout alleles for most genes expressed in ES cells. We used an adaptation of a recently published site-specific recombination strategy termed FlEx (flip-excision) [11]. FlEx uses pairs of inversely oriented heterotypic recombinase target sequences (RTs) such as loxP and lox511 or frt and F3. When inserted upstream and downstream of a gene-trap cassette, Cre or FLPe recombinases invert the cassette and place two homotypic RTs near to each other in a direct orientation. Recombination between this pair of directly repeated RTs excises one of the other heterotypic RTs, thereby ‘locking’ the recombination product against re-inversion to the original orientation. To exploit these features, we equipped retroviral gene-trap vectors encoding a beta-galactosidase/neomycinphosphotransferase (ßgeo) [12] or a CD2 antigen/neomycinphosphotransferase (Ceo) [10] with a double FlEx array consisting of frt/F3 and loxP/lox511 pairs on both sides of the gene-trap cassettes (Figure 1A). Mutagenic insertions of the resulting FlipRosaßgeo and FlipRosaCeo into mouse genes are susceptible to both FLPe and Cre-mediated recombination, which, when delivered in succession, will induce two directional inversions. As exemplified in Figure 1B for FlipRosaßgeo, this will first repair and then re-induce the gene-trap mutation.
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Figure 1: Conditional gene-trap vectors and mechanism of gene inactivation. (A) Schematic representation of the retroviral gene-trap vectors. LTR, long terminal repeat; frt (white triangles) and F3 (black triangles), heterotypic target sequences for the FLPe recombinase; loxP (dark grey triangles) and lox511 (light grey triangles), heterotypic target sequences for the Cre recombinase; SA, splice acceptor; ßgeo, beta-galactosidase/neomycinphopshotransferase fusion gene; pA, bovine growth hormone polyadenylation sequence; TM, human CD2 receptor transmembrane domain. (B) Conditional gene inactivation by an SAßgeopA cassette. The SAßgeopA cassette flanked by recombinase target sites (RTs) in a FlEx configuration is illustrated after integration into an intron of an expressed gene. Transcripts (shown as grey arrows) initiated at the endogenous promoter are spliced from the splice donor (SD) of an endogenous exon (here exon 1) to the SA of the SAßgeopA cassette. Thereby the ßgeo reporter gene is expressed and the endogenous transcript is captured and prematurely terminated at the cassette's pA causing a mutation. In step 1, FLPe inverts the SAßgeopA cassette onto the non-coding strand at either frt (shown) or F3 (not shown) RTs and positions frt and F3 sites between direct repeats of F3 and frt RTs, respectively. By simultaneously excising the heterotypic RTs (step 2), the cassette is locked against re-inversion as the remaining frt and F3 RTs cannot recombine. This reactivates normal splicing between the endogenous splice sites and deletes the SA geopA cassette from the mature transcript, thereby repairing the mutation. Cre-mediated inversion in steps 3 and 4 repositions the SAßgeopA cassette back onto the coding strand and reinduces the mutation. Note that the recombination products of steps 1 and 3 are unstable and thus exist only transiently.
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After extensive
in vitro validation studies, the FlipRosaßgeo
and FlipRosaCeo vectors have been used by the German Gene Trap
Consortium to assemble the largest library of ES cells harbouring
conditional mutations in single genes yet assembled, presently
totalling 1724 genes.
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MULTIPURPOSE ALLELES
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The conditional gene-trap resource is freely available for immediate
mutant mouse production with either germ line or somatic mutations
in single genes. Moreover, by introducing heterologous recombinase
target sites into the genome, the trapped ES cell lines are
amenable to a large variety of post-insertional modifications
by recombinase-mediated cassette exchange (RMCE) [
13]. Examples
include replacing the gene-trap cassettes with inducible Cre
recombinase genes to expand the Cre-zoo, or point mutated minigenes
to study point mutations. Further options are the induction
of gain-of-function mutations or the ablation of specific cell
lineages by inserting gain-of-function cassettes or toxin genes
[
14], respectively.
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THE EUCOMM PROJECT
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Analysis of the mouse and human genomes has resulted in the
identification of
28 000 genes as well as hundreds of thousands
of conserved non-coding regions. Now that the human and mouse
sequences are available, attention has turned to the next phase
of the project, elucidation of gene function in the context
of the entire organism. Gene trapping and targeting allow systematic,
cost-effective and high-throughput generation of mutations in
ES cells. ES cells can be easily stored, distributed and converted
into mice. The value of the information gained from the analysis
of mouse mutants is widely acknowledged and therefore, it was
agreed by the international scientific community that the time
is ripe for a worldwide concerted action to perform saturation
mutagenesis of the mouse genome using gene-targeting and gene-trapping
approaches [
1,
2]. Such an enormous task can only be achieved
by an internationally coordinated effort to systematically mutate
all genes for the benefit of the scientific community. An organized
systematic assault reduces redundancy, facilitates standardization,
enables technical developments to be implemented globally and
thereby minimizes the costs compared with uncoordinated efforts.
Moreover, organized efforts enable data and resources to be
held in repositories, which enforces standard data formats and
guarantees rapid availability of resources (vectors, ES cells
and mutant mice) without restrictions.
The European Conditional Mouse Mutagenesis project (EUCOMM) has been initiated to contribute to this ambitious goal. The project is built on exceptionally strong European expertise in mouse molecular genetics, genomics and bioinformatics. It is jointly coordinated by the National Research Center for Environment and Health (GSF, Munich, Germany) and the Wellcome Trust Sanger Institute (Hinxton, UK) and involves besides the coordinating institutions the Department of Molecular Hematology, University of Frankfurt Medical School (Frankfurt, Germany), Max-Planck-Institute of Molecular Genetics (Berlin, Germany), University of Dresden (Dresden, Germany), Gene Bridges (Dresden, Germany), Institute Clinique de la Souris (Strasbourg, France), European Molecular Biology Laboratories (EMBL, Monterotondo, Italy), Medical Research Council (Harwell, UK), National Research Council (Monterotondo, Italy) and the German Resource Center of Genome Research (RZPD, Heidelberg, Germany).
In close collaboration with similar programmes initiated in the US (Knockout Mouse Project, KOMP [1]) and Canada (North American Conditional Mouse Mutagenesis Project, NorCOMM), EUCOMM will complement high-throughput gene-targeting resources with ready-to-use conditional mutations in all genes throughout the mouse genome. As a first step in this direction, the publically available gene trap lines have been recently centralized on the International Gene Trap Consortium's (IGTC) web page (http://www.igtc.ac.org/) [15].
Key Points- This article summarizes the current developments in the generation of a mutant mouse database within the EUCOMM.
- Conditional gene traps have been implemented and will be used to create mutant mouse ES cell lines for every gene of the mouse genome.
- The technique of conditional gene traps uses the recently published FlEx system to allow recombinase-mediated unidirectional inversions.
- The generated clones will be amenable to a large variety of post-insertional modifications.
- All the generated clones will be freely available from the IGTC web page (http://www.igtc.ac.org/).
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Acknowledgements
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I thank Prof. Harald von Melchner and all members of the German
Gene Trap Consortium for fruitful discussions and continuous
support.
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FOOTNOTES
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Frank Schnütgen leads the vector development group in Harald
von Melchner's laboratory and is a member of the German Gene
Trap Consortium.
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References
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