Retargeting of retroviral integration sites for the predictable expression of transgenes and the analysis of cis-acting sequences.
The transcriptional activity of transgenes in eukaryotic host cells critically depends on the sites of their integration where it is modulated by interactions between the promoter and surrounding chromatin structures. Retroviruses integrate their genome into chromosomal loci favoring expression from one long-terminal repeat (LTR). We have therefore developed a strategy in which retroviral vectors are provided with "tags", that is, targets for a site-specific recombinase (Flp). Presence of two 48 bp Flp recognition target (FRT) sequences permits the excision of a selection marker whereby the reading frame of a reporter gene (lacZ) is restored and beta-galactosidase activity can be monitored to characterize the integration site regarding the level and stability of expression. The location of the remaining FRT tag permits the subsequent Flp-mediated insertion of a truncated selection marker which is then expressed from the LTR. This step represents an authentic site-specific recombination event which can be demonstrated by a number of criteria, among these its reversibility in the presence of Flp activity. Thereby the "expression trap" principle permits the efficient recovery of stable insertion events, and if a gene of interest is linked to the truncated marker, the established properties of a given genomic site can be utilized for transcription studies or for the generation of highly expressing clones, even for biotechnological purposes.
GBF, National Research Center for Biotechnology, Braunschweig, Germany.
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Schübeler
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Maass
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Bode
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D005167:Factor VII / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D019076:Transgenes; D016662:Virus Integration / Q000235:genetics*
D005167:Factor VII