Nucleic Acids Research, Vol 27, Issue 24 e35-e35, Copyright © 1999 by Oxford University Press
Y Ishida and P Leder
Gene trapping is a form of insertional mutagenesis that causes disruption
of gene function. Herewe 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.
ARTICLES
RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells
Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA
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