RESUMO
It has been previously demonstrated that the wild type integrase (Int) protein of coliphage HK022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. In the present report it is shown that Int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. These include integrative (attP x attB) as well as excisive (attL x attR) reactions each in two configurations. In the cis configuration both sites are on the same chromosome, in the trans configuration one site is on a chromosome and the other on an episome. The reactions in cis were observed without any selection force, using the green fluorescent protein (GFP) as a reporter. The reactions in trans could be detected only when a selection force was applied, using the hygromycin-resistant (Hyg(R)) phenotype as a selective marker. All reactions were catalyzed without the need to supply any of the accessory proteins that are required by Int in its Escherichia coli host. The versatility of the att sites may be an advantage in the utilization of Int to integrate plasmid DNA into the genome, followed by a partial exclusion of the integrated plasmid.
Assuntos
Bacteriófago HK022/enzimologia , Genoma Humano/genética , Integrases/metabolismo , Recombinação Genética/genética , Southern Blotting , Linhagem Celular , DNA/genética , DNA/metabolismo , Humanos , Modelos Genéticos , Plasmídeos/genéticaRESUMO
The Integrase (Int) site-specific recombinase of coliphage HK022 catalyzes integrative and excisive DNA recombination between two attachment (att) sites in human cells without the need to supply the accessory proteins Integration Host Factor (IHF) and Excisionase (Xis). Previous work has shown that under these conditions, reactions in cis, i.e. both att sites are located on the same chromosome, can be detected without selection. However, recombination in trans, i.e. one att site positioned on a chromosome and the other on an episomal vector, was detected only after selection. Here we show that optimization of the int-HK022 gene for human codon usage according to the GeneOptimizer software algorithm, as well as addition of accessory proteins IHF and Xis improve the recombination efficiencies in human cells, such that recombinants in a trans reaction could be detected without selection.