390. Phage c31 Integrase Induces Chromosomal Aberrations in Primary Human Fibroblasts

• DOI: 10.1016/j.ymthe.2006.08.451
• Publication Date: 2010-08-24T12:47:46Z
• Authors: Jian Liu; Iben Jeppesen; Kate Nielsen; Thomas G. Jensen
• Keywords: Integrases

Phage c31 integrase is a recombinase with the potential to perform site-specific integration into host chromosome. The enzyme mediates recombination between two different short sequences of DNA, attP and attB recognition sites. In mouse human genomes, phage plasmids bearing an site partial sequence identity attP, called pseudo Based on identification sites in genome it was estimated that total number 102 103. integrase-mediated gene delivery under investigation as novel tool for therapy. order investigate safety integration, we have studied effects prolonged expression primary fibroblasts, derived from spontanous abortion. Embryonic cells were used due their high proliferative genomic stability.Primary fibroblasts transfected pBabepuro or pBabepuroatt, containing 285-base pair sequence, without plasmid pCMVInt expressing integrase. co-transfection experiments performed 3:1 molar excess compared pBabepuro/pBabepuroatt increase likelihood integrated puromycin resistant cells. After selection, besides cytogenetic analysis, DNA RNA also isolated analysed using primers specific most common (the aatP A chromosome 8).Cells pBabepuroatt alone had normal karyotypes. contrast highly abnormal karyotypes found plasmids. No obvious pattern involving chromosomes observed, previously published not involved more frequently than other aberrations. Only 1 14 identified breakpoints matched localisation site. Analysis puromycin-selected clones likewise showed reproduced another embryo, again, exclusively gene.Thus shown induces rearrangement raising serious concerns about possible clinical use. Another member family recombinases, Cre recombinase, has ability cause widespread damage when expressed at levels. Here pseudo-sites suspected act target give rise mutagenic background. Since transgenic mice Drosophila been generated no adverse consequences, further work required understand mechanisms behind mediated instability. stability. Primary 8). Cells gene. Thus