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 143

DNAs are labeled with biotin-16-dUTP and digoxigenin-11-dUTP,
respectively, by nick translation kit (Roche, Hague Road, IN,
USA) according to the manufacturer's protocol for use as probes.
After hybridization, the slides are washed in 50% formamide/2 ×
SSC at 37°C and in 1 × SSC at room temperature for 20 min each.
Detection of the probe signals is performed with Cy3-labeled
streptavidin (3 µg/ml) and Cy5-labeled anti-digoxigenin (3 µg/
ml) for biotin-labeled and digoxigenin-labeled probes, respec-
tively. FISH images are captured by the CW4000 FISH applica-
tion program of Leica Microsystems Imaging Solution Ltd. with
a cooled CCD camera mounted on a Leica DMRA2 microscope.
Multicolor FISH (M-FISH) with human chromosome-specific
paints is performed after hybridization with biotin-labeled
ABCG2-pcDNA5/FRT for assignment of the chromosomes
where the transgenes are located. The M-FISH images are cap-
tured and merged with the images of the hybridization signals of
the ABCG2-pcDNA5/FRT on the same metaphase spreads by
the CW4000 FISH application program.

It is important to examine whether the genomic DNA-integrated
ABCG2 cDNA is transcribed into mRNA. The transcript can be
detected by conventional RT-PCR or real time-PCR methods
(for ABCG2 results see Note 3). Total RNA is extracted from
cultured cells with (for example with the NucleoSpin ® RNA II
kit). cDNA is prepared from the extracted RNA in a reverse tran-
scriptase reaction with SuperScript II RT and random hexamers
according to the manufacturer's instructions. The mRNA levels
of ABCG2 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) are determined by PCR in an iCycler TM thermal cycler
(BIO-RAD, Hercules, CA, USA) with the ABCG2 and GAPDH
primer sets. The PCR reaction consists of hot-start incubation at
94°C for 2 min and 30 cycles of 94°C for 30 s, 59°C for 30 s, and
72°C for 30 s. After the PCR, products were separated by agarose
gel electrophoresis and detected with ethidium bromide under
UV light. The RNA levels of ABCG2 and GAPDH can be quan-
titatively determined by using the 7500 Fast Real Time-PCR
System with TaqMan ® Fast Universal Master Mix, and TaqMan ®
probes. The expression levels of ABCG2 should be normalized
against those of GAPDH.

3.3.2. Quantitative Analysis
of ABCG2 mRNA
in Flp-In-293 Cells by
RT-PCR

The ABCG2 protein expressed in Flp-In-293 cells can be detected
by immunoblotting with BXP-21 (ALEXIS Co., Lausen,
Switzerland), a specific antibody to human ABCG2 (see Note 4).
The cells are rinsed with ice-cold PBS and subsequently treated
with lysis buffer containing 50 mM Tris/HCl (pH 7.4), 1% (w/v)
Triton X-100, 1 mM DTT, and a protease inhibitor cocktail
(Roche Ltd., Mannheim, Germany). The samples are homoge-
nized by passage through a 27-gauge needle and then centrifuged

3.3.3. Detection
of N-Linked Glycosylation
and Disulfide Formation
of ABCG2
by Immunoblotting