Genetic engineering of herpes simplex virus and vector genomes carrying loxP sites in cells expressing Cre recombinase.
The prokaryotic Cre-loxP recombination system is a powerful tool that enables in vitro and in vivo site-specific manipulations of the genome of eukaryotic cells as well as of DNA viruses and their derived vectors. This system, however, has not yet been exploited in the context of herpes simplex virus type 1 (HSV-1) infected cells, perhaps because this virus encodes several functions that induce a strong shutoff of cellular protein synthesis, a fact that could preclude expression of cellular-encoded Cre recombinase. In the present study, we show that efficient site-specific recombination can take place in cell lines expressing Cre, even in the context of HSV-1 infection, as evidenced by the engineering of an HSV-1 recombinant virus and several viral vectors carrying one or two loxP sequences. More precisely, we have used this system to induce an irreversible switch in the expression of a viral complex transcription unit encoding two different open reading frames and allowing consecutive expression of two reporter genes. Furthermore Cre recombinations were also used to induce the decatenation of the genomic concatemers harbored by amplicon particles upon infection of cells under nonreplicative conditions, thus enabling the rescue of many independent plasmids corresponding to the original amplicon plasmid used to generate the vectors. Thus the Cre-loxP recombination system can successfully be used for engineering the genome of HSV-1 or HSV-1-based vectors in cultured cells.
Logvinoff
C
C
Centre de Génétique Moléculaire et Cellulaire, Centre National de la Recherche Scientifique-UMR 5534, Université Claude Bernard Lyon 1, 43 boulevard du 11 novembre 1918, Villeurbanne Cedex, 69622, France.
Epstein
A L
AL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D016679:Genome, Viral; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D019426:Integrases / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014709:Vero Cells; D014764:Viral Proteins
D017931:DNA Primers; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases