Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  G van den Engh,et al.  Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes. , 1986, Cold Spring Harbor symposia on quantitative biology.

[3]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[4]  W. C. Forrester,et 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. , 1992, Genes & development.

[5]  Alexandra L. Joyner,et al.  Gene targeting: a practical approach. , 1993 .

[6]  J. Bode,et al.  Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. , 1997, Biochemistry.

[7]  P. Gruss,et al.  Evidence for the stochastic integration of gene trap vectors into the mouse germline. , 1997, Nucleic acids research.

[8]  H. Ruley,et al.  Functional genomics in mice by tagged sequence mutagenesis , 1997, Nature Genetics.

[9]  P. Gruss,et al.  Efficient gene trap screening for novel developmental genes using IRES beta geo vector and in vitro preselection. , 1998, Experimental cell research.

[10]  M. Groudine,et al.  DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs. , 1998, Biochemistry.

[11]  P. Leder,et al.  RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells. , 1999, Nucleic acids research.

[12]  K. Yamamura,et al.  Exchangeable gene trap using the Cre/mutated lox system. , 1999, Cellular and molecular biology.

[13]  M. Metzker,et al.  Optimization of the helper-dependent adenovirus system for production and potency in vivo. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. Nagy,et al.  Gene‐trap‐based target site for Cre‐mediated transgenic insertion , 2000, Genesis.

[15]  A. Joyner,et al.  Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. , 2000, BioTechniques.

[16]  P. Gruss,et al.  Mice deficient for spermatid perinuclear RNA-binding protein show neurologic, spermatogenic, and sperm morphological abnormalities. , 2001, Developmental biology.

[17]  K. Rajewsky,et al.  New variants of inducible Cre recombinase: a novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and an expanded range of inducibility. , 2001, Nucleic acids research.

[18]  J. Minna,et al.  Genomic Organization and Mapping of the Gene Encoding the PP2A B56γ Regulatory Subunit , 2002 .

[19]  J. Minna,et al.  Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit. , 2002, Genomics.

[20]  Franz Vauti,et al.  A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Peter Vogel,et al.  Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  N. Perrimon,et al.  Tailoring the genome: the power of genetic approaches , 2003, Nature Genetics.

[23]  Conrad C. Huang,et al.  BayGenomics: a resource of insertional mutations in mouse embryonic stem cells , 2003, Nucleic Acids Res..

[24]  Shiaoching Gong,et al.  A gene expression atlas of the central nervous system based on bacterial artificial chromosomes , 2003, Nature.