The FRT integration vec tor pcDNA5/FRT/TO DsRed consists of the red fluorescent protein DsRed as GOI, whereas the attP inte gration vector pINT PuroDDEYFP is made up of as GOI the yellow fluorescent protein EYFP linked for the destabilizing domain. In the 1st round of transfection, the DsRed gene was launched applying the FLP recombi nase. The obtained cell lines from just about every clone were employed to get a 2nd round of transfection applying the EYFP con taining attP integration vector and the FC31 integrase. The resulting cell lines had been resistant to hygromycin and puromycin and have consequently potentially integrated the 2 transgenes. To check whether or not the two transgenes were existing and conditionally energetic, we handled all cell lines with doxycy cline, a reasonably secure tetracycline derivative, or Shld1 and monitored red or yellow fluorescence.
In all cell lines derived from double docking cell lines 12, 16, and 19, red fluorescence was particularly induced by doxycycline and yellow fluorescence by Shld1 in more than 90% on the cells. As an example, a cell line derived selleck chemical from double docking cell line 19 is proven in Figure two. Plainly, there was no cross response between the 2 conditional systems, and the two genes is often induced in parallel. Up coming, we quantified the degree of induction by western blot analyses. Doxycycline treatment method triggered an about 50 fold greater DsRed expression in the cell lines sixteen one 3 and 12 one 1, and about 20 fold induction in 19 three 1. When Shld1 was offered towards the cells, the DD EYFP protein was induced about 30 fold in sixteen one three, twenty fold in twelve one 1, and twenty fold in 19 3 1.
Of note, within the cell lines 16 one three and 12 1 1 the ECFP Neo fusion protein was nevertheless expressed. We presume that in these cell lines there is at the very least a single un recombined copy of the attP docking web site current, even though the transgene was integrated. In contrast, the cell line 19 three one lacks ECFP Neo expression arguing for recombination selleck at a one of a kind attP docking web page. Thus, the cell line 19, we refer to as HEK attP/FRT cell line, is finest suited to realize secure integration of two distinct trans genes with independent conditional activation of each transgene. Independent integration of two inducible HNF4a proteins To confirm that this double conditional technique could also be made use of to express genes interfering with cell cycle progres sion we introduced the transcription component HNF4a splice variant 2. which has been shown to inhibit HEK293 cell multiplication into each docking web-site. In the initial round of transfection, the doxycycline inducible HNF4a2 sequence was launched to the FRT site in the HEK attP/FRT cells working with the FLP recombinase. Site precise integration was verified by damaging lacZ staining in three independent cell lines.