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 139

was originally isolated from the S. cerevisiae 2-mm plasmid ( 43,
44 ) and encodes a site-specific recombinase that is a member
of the integrase family of recombinases ( 45 ). The Flp recom-
binase mediates a site-specific recombination reaction between
interacting DNA molecules via the pairing of interacting FRT
sites ( 29, 46 ). The native FLP gene encodes a protein of 423
amino acids with a calculated molecular mass of 49 kDa. The
FLP gene expressed from pOG44 encodes a temperature-
sensitive Flp recombinase, which carries a point mutation (flp-
F70L) that results in a change in amino acid 70 from Phe to
Leu ( 47 ). The flp-F70L protein expressed from pOG44 exhib-
its increased thermo-stability at 37°C in mammalian cells when
compared with the native Flp recombinase ( 47 ).
4. Dulbecco's modified Eagle's medium (D-MEM; Nacalai
Tesque, Inc., Kyoto, Japan).
5. 10% (v/v) heat-inactivated fetal calf serum (FCS; Dainippon
Pharmaceuticals, Osaka, Japan).
6. l l-Glutamine (Wako Pure Chemical Industries, Ltd., Osaka,
Japan), 2 mM.
7. Penicillin (100 U/ml) (Invitrogen, Carlsbad, CA, USA).
8. Streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA, USA).
9. Zeocin, 100 mg/ml (Invitrogen, Carlsbad, CA, USA).
10. Hygromycin B, 100 mg/ml (Invitrogen, Carlsbad, CA, USA).
11. Amphotericin B, 250 ng/ml (Invitrogen, Carlsbad, CA, USA).
12. 12- and 24-well culture plates (Becton Dickinson and
Company, Franklin Lakes, NJ).
13. CO 2 incubator.
14. Trypan Blue dye (Nacalai Tesque, Inc., Kyoto, Japan).
15. Lipofectamine-2000 (Invitrogen, Carlsbad, CA, USA).

2.2. Validation
of the System

1. Biotin 16 dUTP (Roche, Hague Road, IN, USA).
2. Digoxigenin-11-dUTP (Roche, Hague Road, IN, USA).
3. SSC (Sodium Citrate & Sodium Chloride, Wako Chemicals,
Osaka, Japan).
4. Formamide (Wako Chemicals, Osaka, Japan).
5. Cy3-labeled streptavidin (Amersham Pharmacia Biotech,
Uppsala, Sweden).
6. Cy5-labeled anti-digoxigenin (Roche, Hague Road, IN,
USA).
7. Cooled CCD camera mounted on a microscope (e.g., Leica
DMRA2).
8. Imaging software (e.g., CW4000 FISH application program
of Leica Microsystems Imaging Solution Ltd.).

2.2.1. FISH Analysis