Abstract
Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines.
Highlights
Gene targeting is a powerful technology to explore gene functions in eukaryotes by allowing the manipulation of endogenous genes via homologous recombination (HR)
This targeting vector was created based on an associated virus (AAV) backbone, as AAVbased targeting vectors reportedly achieve more than 1,000-times higher gene targeting frequencies than conventional plasmidbased targeting vectors [6], and have been employed in many recent studies to overcome low gene targeting efficiencies in human somatic cell lines [5,20,21,22,23,24,25,26,27,28]
We developed a molecular system that exploits a unique property of the phosphatidylinositol glycan anchor biosynthesis class A (PIGA) gene to evaluate gene targeting efficiencies in human somatic cell lines
Summary
Gene targeting is a powerful technology to explore gene functions in eukaryotes by allowing the manipulation of endogenous genes via homologous recombination (HR). Several significant improvements in gene targeting technology have been made in previous studies, including the use of adenoassociated virus (AAV)-based targeting vectors [6,7], a promotertrap strategy with the aid of internal ribosomal entry sites (IRES) [8], and a systematic PCR screening method for gene-targeted cell clones [9]. Despite these improvements, gene targeting in most of human somatic cell lines remains to be difficult probably because of low HR efficiencies as demonstrated in murine somatic cells [10]. Monitoring the frequency of homologous versus random integration (H/R ratio) of targeting vectors is informative, as this ratio is a major determinant of difficulty in conducting gene targeting
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