RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells.
Gene trapping is a form of insertional mutagenesis that causes disruption of gene function. Here we report the construction and extensive examination of a versatile retrovirus vector, RET (removable exon trap). The RET vector uses an improved poly A-trap strategy for the efficient identification of functional genes regardless of their expression status in target cells. A combination of a potentially very strong splice acceptor and an effective polyadenylation signal assures the complete disruption of the function of trapped genes. Inclusion of a promoterless GFP cDNA in the RET vector allows the expression pattern of the trapped gene to be easily monitored in living cells. Finally, because of loxP-containing LTRs at both ends, the integrated proviruses can be removed from the genome of infected cells by Cre-mediated homologous recombination. Hence, it is possible to attribute the mutant phenotype of gene-trapped cells directly to RET integration by inducing phenotypic reversion after provirus excision. The RET system can be used in conjunction with cell lines with functional heterozygosity, embryonic stem cells, lineage-committed cell lines that differentiate in response to specific inducing factors and other responsive cell lines that can be selected by virtue of their induced green fluorescence protein expression.
Ishida
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Y
Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA.
Leder
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P
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D005069:Evaluation Studies as Topic; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D019426:Integrases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D011061:Poly A / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014764:Viral Proteins; D016662:Virus Integration
D008164:Luminescent Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D011061:Poly A; C045073:Cre recombinase; D019426:Integrases