U-2 OS expression lines were generated by transfection of U-2 OS T-Rex Flp-In cells with pOG44 Flp-Recombinase plasmid (Thermo Fisher Scientific) and pcDNA5/FRT/TO plasmid at a 9:1 ratio, followed by selection in 500 μg/mL hygromycin. NCI-H1703 and NCI-H446 expression lines were generated by infection with pLenti CMV TetR Blast virus (716-1) (Addgene plasmid #17492) in the presence of 8 μg/mL polybrene (Sigma-Aldrich), followed by selection in medium containing 2 μg/mL blasticidin for NCI-H1703 cells and 0.5 μg/mL blasticidin for NCI-H446 cells. TetR cells were subsequently infected with pLenti CMV/TO Hygro DEST virus (670-1) (Addgene plasmid #17293) containing the FSP1-GFP construct and were selected in medium containing 250 μg/mL hygromycin. FSP1-GFP-expressing cells were enriched by fluorescence-activated cell sorting of the GFP-positive populations.
Pog44 flp recombinase plasmid
Manufactured by Thermo Fisher Scientific
The POG44 Flp-Recombinase plasmid is a genetic construct designed to facilitate site-specific recombination. It encodes the Flp recombinase enzyme, which catalyzes the recombination of DNA sequences containing Flp recognition target (FRT) sites. The plasmid is a tool commonly used in molecular biology research, but its specific intended use is not provided.
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2 protocols using pog44 flp recombinase plasmid
1
Generation of Inducible Cell Lines
2
Generation of Inducible Cell Lines
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U-2 OS expression lines were generated by transfection of U-2 OS T-Rex Flp-In cells with pOG44 Flp-Recombinase plasmid (Thermo Fisher Scientific) and pcDNA5/FRT/TO plasmid at a 9:1 ratio, followed by selection in 500 μg/mL hygromycin. NCI-H1703 and NCI-H446 expression lines were generated by infection with pLenti CMV TetR Blast virus (716-1) (Addgene plasmid #17492) in the presence of 8 μg/mL polybrene (Sigma-Aldrich), followed by selection in medium containing 2 μg/mL blasticidin for NCI-H1703 cells and 0.5 μg/mL blasticidin for NCI-H446 cells. TetR cells were subsequently infected with pLenti CMV/TO Hygro DEST virus (670-1) (Addgene plasmid #17293) containing the FSP1-GFP construct and were selected in medium containing 250 μg/mL hygromycin. FSP1-GFP-expressing cells were enriched by fluorescence-activated cell sorting of the GFP-positive populations.
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