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 135

and six transmembrane domains. Human ABCG2 exists in the
plasma membrane as a homodimer bound through disulfide-
bonded cysteine residues ( 15-19 ). The expression level of ABCG2
is regulated by both synthesis and degradation of the protein. By
using Flp-In-293 cells, we have recently demonstrated that the for-
mation of an intramolecular disulfide bond between Cys592 and
Cys608 and N -linked glycosylation at Asn596 are critical check
points for the stability and degradation of the de novo synthesized
ABCG2 protein ( 20, 21 ). Furthermore, certain non-synonymous
single nucleotide polymorphisms (SNPs), such as Q141K, F208S,
and S441N, were also found to greatly affect the stability of ABCG2
in the ER and to enhance the protein degradation rate via ubiquit-
ination and proteasomal proteolysis in Flp-In-293 cells ( 22-27 )
(Fig. 1 ). In this regard, we could successfully analyze the quality
control of ABCG2 in the ER, namely, posttranslational modifica-
tions (intra- and inter-molecular disulfide bond formation and
N -linked glycosylation) as well as ubiquitin-mediated proteasomal
degradation of the ABC transporter protein ( 20, 21 ).
On the basis of our recent studies on the quality control of
ABCG2 in the ER ( 19-27 ), we describe our methods for the
production of Flp-In-293 cells with targeted integration of gene
variants of the human ABC transporter for stable and regulated
expression with the Flp recombinase system. The Flp-In-293 cell
line appears to carry biologically important components required
for post-translational modifications ( N -linked glycosylation, dis-
ulfide bond formation, ubiquitination, etc.) of de novo synthe-
sized proteins and also the machinery for protein degradation via
endosome/lysosome or proteasome pathways. Taken together, it
is concluded that the Flp recombinase system provides a useful
tool to quantitatively analyze the protein stability and degrada-
tion of misfolded proteins. Expression of ABCG2 is used as an
example and methods to validate successful integration, expres-
sion, and subcellular localization of this protein are described,
which can be adapted to other proteins that can be expressed in
the same way.

2. Materials

2.1. Flp-In TM System

1. Flp-In cell lines:
(a) Flp recombinase-mediated site-specific integration and
gene expression in mammalian cells was originally devel-
oped by S. O'Gorman, D. T. Fox, and G. M. Wahl at the
Gene Expression Laboratory, The Salk Institute for
Biological Studies, La Jolla, CA, in 1991 ( 28 ). The Flp
recombinase system allows us to integrate one single copy
of cDNA into the genomic DNA at a specific genome