Design of the integration systems
To allow site specific integration of two different transgenes we used two independent systems. The first is based on FLP recombinase mediated recombination of FRT sites (Figure 1A). By using hygromycin resistance, recombined cell lines can be selected. Moreover, loss of lacZ reporter activity monitors specific recombination with the FRT integration vector. Conditional transcriptional control of the transgene is achieved by applying the tetracycline inducible system. This system is well established and commercially available (Flp-In T-Rex™, Invitrogen). As a second and independent system we developed ΦC31 integrase mediated recombination of attP and attB sites (Figure 1B). To allow site directed integration of any gene-of-interest (GOI) using the ΦC31 integrase, we designed the docking construct pDOCKING-Neo containing the attP recognition site. This site is linked in frame to an ECFP-neomycin resistance fusion protein as selection marker. As the attP site is placed downstream of the start codon for ECFP-Neo, this marker will be inactivated upon specific recombination with the corresponding attB sequence. The incoming attB integration vector contains the attB site fused to a promoter- and ATG-less puromycin resistance gene, which is thus activated upon specific recombination with the attP sequence of the docking site. The GOI is placed downstream of the puromycin resistance sequence. In all experiments we used the CMV promoter and fused the GOI to the L106P mutant of the human FKBP12 protein (destabilizing domain, DD) to regulate the expression on the level of protein stability by Shld1 [7].
Generation of double docking HEK293 cell lines
The commercially available Flp-In T-Rex™ HEK293 cells contain a stably integrated single copy FRT docking site for FLP mediated integration. Using this cell line we introduced in addition for ΦC31 mediated transgene integration the newly generated attP docking site that encodes an ECFP-Neo fusion gene. After selection with G418 we selected 24 single clones with blue fluorescence. To determine the number of integrated transgene copies we performed real-time PCR on genomic DNA and selected the three double docking cell lines 12, 16, and 19 with apparent single copy integration of the docking site for further experiments.
Independent integration of inducible fluorescent proteins into different double docking HEK293 cell lines
To test whether independent integration as well as independent conditional activation of two different GOI can be achieved in the double docking cell lines we generated two different integration vectors. The FRT integration vector pcDNA5/FRT/TO-DsRed contains the red fluorescent protein DsRed as GOI (Figure 1A), whereas the attP integration vector pINT-PuroDDEYFP contains as GOI the yellow fluorescent protein EYFP linked to the destabilizing domain (DD in Figure 1B). In a first round of transfection, the DsRed gene was introduced using the FLP recombinase. The obtained cell lines from each clone were used for a second round of transfection using the EYFP containing attP integration vector and the ΦC31 integrase. The resulting cell lines were resistant to hygromycin and puromycin and have thus potentially integrated the two transgenes.
To test whether the two transgenes were present and conditionally active, we treated all cell lines with doxycycline, a relatively stable tetracycline derivative, or Shld1 and monitored red or yellow fluorescence (Figure 2). In all cell lines derived from double docking cell lines 12, 16, and 19, red fluorescence was specifically induced by doxycycline and yellow fluorescence by Shld1 in more than 90% of the cells. As an example, a cell line derived from double docking cell line 19 is shown in Figure 2. Clearly, there was no cross-reaction between the two conditional systems, and both genes can be induced in parallel. Next, we quantified the level of induction by western blot analyses. Doxycycline treatment caused an about 50-fold increased DsRed expression in the cell lines 16-1-3 (Figure 3A) and 12-1-1 (not shown), and about 20-fold induction in 19-3-1 (figure 3B). When Shld1 was given to the cells, the DD-EYFP protein was induced about 30-fold in 16-1-3 (Figure 3A), 20-fold in 12-1-1 (not shown), and 20-fold in 19-3-1 (Figure 3B). Of note, in the cell lines 16-1-3 (Figure 3A) and 12-1-1 (not shown) the ECFP-Neo fusion protein was still expressed. We assume that in these cell lines there is at least one un-recombined copy of the attP docking site present, although the transgene was integrated. In contrast, the cell line 19-3-1 lacks ECFP-Neo expression arguing for recombination at a unique attP docking site. Thus, the cell line 19, we refer to as HEK-attP/FRT cell line, is best suited to achieve stable integration of two distinct transgenes with independent conditional activation of each transgene.
Independent integration of two inducible HNF4α proteins
To verify that this double conditional system can also be used to express genes interfering with cell cycle progression we introduced the transcription factor HNF4α splice variant 2. (HNF4α2) that has been shown to inhibit HEK293 cell multiplication [3, 4] into each docking site. In a first round of transfection, the doxycycline inducible HNF4α2 sequence was introduced into the FRT site of the HEK-attP/FRT cells using the FLP recombinase. Site specific integration was verified by negative lacZ staining in three independent cell lines. One cell line 19-2 was used for a second round of transfection to integrate Shld1 inducible HNF4α2 into the attP docking site using ΦC31 integrase. The resulting four cell lines (19-2-2, 19-2-6, 19-2-4 and 19-2-5) were resistant to hygromycin and puromycin and have thus potentially integrated the two transgenes. To verify site specific integration into the attP docking site we screened for loss of ECFP-Neo expression, which is inactivated upon recombination (compare Figure 1B). This loss of ECFP-Neo expression was validated by a western blot using a monoclonal anti-GFP antibody also detecting ECFP (Figure 4A). The parent cell line 19-2 shows expression of the 61 kDa ECFP-Neo fusion protein, whereas no expression was detectable in the cell lines 19-2-2 and 19-2-6 arguing for specific integration of the HNF4α transgene in these cells. In contrast, the cell lines 19-2-4 and 19-2-5 expressed an about 27 kDa protein recognized by the anti-GFP antibody. In these two later cell lines the attP docking site was possibly rearranged by illegitimate recombination resulting in a truncated ECFP-Neo protein. We then tested by western blot analyses whether HNF4α could be induced in these cell lines by doxycycline or Shld1. As the doxycycline inducible HNF4α transgene contains a myc tag, we could differentiate between the doxycycline and Shld1 inducible proteins. In all four cell lines the myc-tagged HNF4α transgene could be induced specifically by doxycycline to a most similar level (Figure 4B, lower panel). However, when treated with Shld1 the cell lines 19-2-2 and 19-2-6 showed activation of HNF4α, whereas the cell lines 19-2-4 and 19-2-5 lacked HNF4α expression despite of puromycin resistance indicating ΦC31 mediated integration of the transgene. The ability to induce HNF4α by Shld1 in the cell lines 19-2-2 and 19-2-4 correlates with the loss of ECFP-Neo expression indicating specific integration in these two cell lines. In contrast, the cell lines 19-2-4 and 19-2-5 still expressing a truncated ECFP-Neo protein were not inducible by Shld1.
To address the effect of both HNF4α transgenes on cell cycle progression we treated the cell lines 19-2-2 and 19-2-6 with doxycycline and/or Shld1 and measured the cell number over five days by the MTS assay. The cell line 19-2-2 showed an about 10-fold increase of cell mass until day 5 (Figure 5A). However, when doxycycline or Shld1 was given cell multiplication was significantly reduced by 5-fold or 6-fold, respectively. Significantly, administration of doxycycline and Shld1 in parallel retained the cell number at about the starting levels pointing to an additive effect of the two independent HNF4α transgenes. MTS assay of the 19-2-6 cell line (Figure 5B) showed most similar results with a significant reduction of cell multiplication by either doxycycline or Shld1 treatment and a lack of cell multiplication, if doxycyline and Shld1 were applied simultaneously.
In summary, the two HNF4α transgenes could be independently activated by doxycycline or Shld1 and the observed effect on cell multiplication was highly reproducible between the two distinct sites of integration in two individual cell lines. Moreover, the effect of HNF4α appears to be concentration dependent, as activation of the second transgene further delays cell multiplication.