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 138

Fig. 2. (continued) the ABCG2 cDNA and the Flp recombinase expression plasmid pOG44. Flp recombinase mediates
insertion of the expression construct with the ABCG2 cDNA into the genome at the integrated FRT site through site-specific
DNA recombination. In the FISH mapping probed with the ABCG2-pcDNA5/FRT and Multicolor FISH-Human probe set, the
hybridization signal of the transgene was detected in the telomeric region of the short arm of one of chromosomes 12
( yellow arrow ) as overlapped signals of the biotin-labeled ABCG2-pcDNA5/FRT and digoxigenin-labeled pcDNA5/FRT, and
two hybridization signals of the biotin-labeled ABCG2-pcDNA5/FRT (internal ABCG2 genes, white arrows ) were detected
in the q22 region on a pair of chromosomes 4.
additional 13-bp repeat is found in most FRT sites ( 38 ).
While Flp recombinase binds to all three of the 13-bp
repeats, strand cleavage actually occurs at the boundaries
of the 8-bp spacer region ( 37, 38 ).
2. pcDNA5/FRT vector: pcDNA5/FRT (Invitrogen, Carlsbad,
CA, USA) is a 5.1-kb expression vector designed for use with
the Flp-In TM system. This vector contains the following ele-
ments: the human cytomegalovirus (CMV) immediate-early
enhancer/promoter ( 39-41 ), multiple cloning sites with
ten unique restriction sites, which can be used to introduce
the cDNA sequence encoding the protein to be studied (in the
present case ABCG2), the FRT site for Flp recombinase-
mediated integration of the vector into Flp-In host cells; and
hygromycin B-resistance gene for the selection of stable cell
lines ( 42 ).
3. pOG44 vector: pOG44 is a 5.8-kb Flp recombinase expres-
sion vector (Invitrogen, Carlsbad, CA, USA). The FLP gene