Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells.

Vol. 40, No. 4 (2006) BioTechniques 1 Supplementary Figure S1. Structure of constructs used and representation of FRT locus. (A) Plasmids used to detect the Bxb1 integrase-mediated sitespecific recombination. CMVp, SV40t, Luc, stop, and Hygro are cytomegalovirus promoter (CMVp), simian virus 40 terminator (SV40t), luciferase gene, transcription termination sequence, and hygromycin resistance gene, respectively. (B) Representation of chromosomal locus before and after the deletion of stop sequence. LacZ:zeo; β-galactosidase-zeocin fusion gene, Puro; puromycin resistance gene. (C) Representation of chromosomal locus before and after the integration of pPURO-attP or pPURO-attB plasmid. Bxb1 integrase expression plasmid pCMV-Bxb1 was prepared by cloning a synthetic gene (codon optimized for human and mouse expression) encoding the 500 amino acid protein (GeneBank accession no. NP_075302) into pDual plasmid (Stratagene, La Jolla, CA, USA) at the unique Eam1104 I restriction enzyme site present between the CMVp and SV40t sequence. Recombination assay plasmid pCMV-attP/attB was constructed in the plasmid backbone of gWizTM Luc (Gene Therapy Systems, San Diego, CA, USA) by inserting a 52-bp double-stranded attP oligonucleotide (5′-GTGGTTTGTCTGGTCAACCACCGCGGTCTCAGTGGTGTACGGTACAAACCCA-3′), a 1296-bp transcriptional termination or stop sequence from plasmid pBS302 (GenBank accession no. U51223, nucleotides 193–1488), and a 46-bp double-stranded attB oligonucleotide (5′-GGCCGGCTTGTCGACG ACGGCGGTCTCCGTCGTCAGGATCATCCGG-3′) between the SalI and NotI restriction enzyme sites present between the CMVp and luciferase gene. The pFRT-attP/attB was prepared by cloning the CMVp-attP-STOP-attB-Luc fragment of pCMV-attP/attB at the SpeI site present upstream of SV40t in pcDNA5/ FRT plasmid (Invitrogen, Carlsbad, CA, USA). pFRT-attB was made by inserting the 46-bp attB oligonucleotide between the NheI and NotI sites, while pFRTattP was made by inserting the 52-bp attP oligonucleotide at the XhoI and ApaI sites in the pcDNA5/FRT plasmid. pPURO-attB was constructed by replacing the FRT, hygromycin gene, and CMVp sequences of pcDNA5/FRT with Bxb1 attB and puromycin gene sequences (BD Biosciences Clontech, Palo Alto, CA, USA), while pPURO-attP was constructed in a similar manner to pPURO-attB, except with Bxb1 attP site. pCMV-Bxb1 CMVp Bxb1 SV40t