Production of Cells with Targeted Integration of Gene Variants of Human ABC Transporter for Stable and Regulated Expression Using the Flp Recombinase System - Protein Misfolding and Cellular Stress in Disease and Aging - page 133

Chapter 9
Production of Cells with Targeted Integration of Gene
Variants of Human ABC Transporter for Stable
and Regulated Expression Using the Flp Recombinase
System
Kanako Wakabayashi-Nakao, Ai Tamura, Shoko Koshiba, Yu Toyoda,
Hiroshi Nakagawa, and Toshihisa Ishikawa
Abstract
The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation
or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. Such
integration, however, of cDNA occurs randomly at unpredictable sites in the host's chromosomal DNA,
and the number of integrated recombinant DNAs is not controllable. To overcome this problem, we
developed the Flp-In method to integrate one single copy of cDNA encoding the human ABC transporter
ABCG2 into FRT-tagged genomic DNA. Examination of more than 20 metaphase spreads for both
fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis revealed that ABCG2
cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in
Flp-In-293 cells. By using those cells, we investigated the effect of genetic polymorphisms and post-
translational modifications of human ABC transporter ABCG2 on the protein expression and degradation.
On the basis of our experience, it has been concluded that the Flp recombinase system provides a useful
tool to quantitatively analyze the protein stability and endoplasmic reticulum (ER)-associated degradation
of proteins like the ABC transporter. This system is also applicable for similar studies of the biogenesis of
other proteins using the secretory pathway as well as proteins with other cellular localizations.
Key words: Flp recombinase, ATP-binding cassette (ABC) transporter, Genetic polymorphisms,
Endoplasmic reticulum-associated degradation (ERAD), Proteasome, Aggresome, N -linked
glycosylation
1. Introduction
The endoplasmic reticulum (ER) is the site of synthesis and
maturation of proteins destined for the plasma membrane, for
the secretory and endocytic organelles, and for secretion ( 1 ).