Simple monitoring of gene targeting efficiency in human somatic cell lines using the PIGA gene.
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.
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Japan.
Sivasundaram
S
Karnan
Yuko
Y
Konishi
Akinobu
A
Ota
Miyuki
M
Takahashi
Lkhagvasuren
L
Damdindorj
Yoshitaka
Y
Hosokawa
Hiroyuki
H
Konishi
D015139:Blotting, Southern; D049109:Cell Proliferation; D005787:Gene Frequency / Q000235:genetics / Q000502:physiology*; D018390:Gene Targeting; D045325:HCT116 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction
D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein