PhiC31 integrase induces a DNA damage response and chromosomal rearrangements in human adult fibroblasts

Jian Liu,Torben Gjetting,Tina Skjørringe,Thomas G Jensen

doi:10.1186/1472-6750-9-31

Jian Liu, Torben Gjetting + Show 2 more

Open Access

https://doi.org/10.1186/1472-6750-9-31

Copy DOI

Abstract

BackgroundPhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations.ResultsWe have studied possible effects of the PhiC31 integrase expression in human adult fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX phosphorylation – a primary event in the response to DNA double-strand-breaks. Transient transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be detected.ConclusionIn primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations.

Highlights

PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy
In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations
Generation of primary human fibroblasts expressing PhiC31 integrase In gene transfer applications, the PhiC31 integrase mediates the integration of plasmids bearing an attB site into sequences with partial sequence identity to attP

Summary

Introduction

PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. PhiC31 integrase mediated integration leads to prolonged gene expression and has been used for correction of disease models [2,3]. It has been reported that PhiC31 integrase can lead to genomic deletions, chromosomal rearrangements and chromosomal instability [4,5,6,7,8]. In mouse cells it was recently shown that the integrase does lead to imprecise deletion of self excision cassettes [9]. PhiC31-mediated integration of plasmid DNA may be accompanied by chromosomal rearrangements in the mammalian host genome with a frequency up to 15% [10].

Methods

Results

Conclusion