This method can help answer key questions in a wide variety of fields requiring conditional and transient inactivation of a gene of interest by reversibly over-expressing a dominant negative version of it. The main advantage of this technique is that it allows only a partial ablation in protein activity, thus, preserving a residual endogenous expression. Although, here we describe the condition inactivation of RB protein, which functions in a multimeric assembly, this methodology can also be applied to monomeric proteins.
Umesh Pyakurel, our laboratory techinician and a coauthor in the manuscript will demonstrate the procedures. To begin, grow NIH3T3 cells, following the vendor's specifications, at 37 degrees Celsius with 5%CO2, using the recommended cell culture medium containing 10%fetal bovine serum. To cotransfect the cells using pTet-Splice and pCMV-Tet3G vectors, first mix two to four microliters of the lipid-based transfection reagent per well, and one milliliter of incomplete DMEM per well in a 15 milliliter tube, and incubate for five minutes at room temperature.
Add two to three micrograms of plasma DNA per well to this transfection reagent's DMEM mix. Mix and incubate for 20 minutes at room temperature. Add one milliliter of the mix containing DNA and the transfection reagent in DMEM to each well of six-well plate with NIH3T3 cells.
After three to four hours of incubation at 37 degrees Celsius with 5%CO2, add one milliliter of complete medium. Then, add 2 microliters of doxycycline stock to each well and mark the plate as plus-doxycycline. Incubate the cells at 37 degrees Celsius with 5%CO2.
To assess the functionality of the TetO-DN-CB-myc6-Rb1 construct in promoting unscheduled cell proliferation, culture HEI-OC1 cells in DMEM containing 10%FBS, under permissive conditions. For cell proliferation studies, count the HEI-OC1 cells using a cell counter. Plate 10, 000 HEI-OC1 cells per well on a 96-well plate in 200 microliters of volume each.
Incubate the cells over night at 33 degrees Celsius with 5%CO2. To induce transgene expression, add one micrograms per milliliter of doxycycline to a subset of transfected HEI-OC1 cells. Use the other subset labeled as minus-doxycycline and the non-transfected HEI-OC1 cells as controls, and incubate the cells as 33 degrees Celsius with 10%CO2.
To assess cell proliferation, 48 hours after transfection, remove the cell culture medium and add 100 microliters of 1x dye binding solution from a cell proliferation kit to each well of the micro-plate. Follow the manufacturer's protocol and incubate for one hour at 37 degrees Celsius. Use a fluorescence microplate reader to measure the fluorescence intensity of each sample.
To further assess cell proliferation using immunocytochemistry, plate the HEI-OC1 cells on a cover glass placed in each well of a 12-well plate in DMEM. Incubate the cells overnight at 33 degrees Celsius. On the following day, cotransfect to the cells in two wells with the pTet-Splice and the pCMV-Tet3G vectors, and then perform the doxycycline treatment on one well as done with the NIH3T3 cells.
Use one untransfected well as a control. To process the cells for Ki-67 labeling, fix them with 4%paraformaldehyde in PBS for 10 minutes at room temperature. After that, wash them three times with ice-cold PBS for five minutes each.
Add 0.25%nonionic detergent in PBS, and incubate for 10 minutes. Repeat the wash three times in PBS for five minutes each. Use 10%serum to block the cells.
In a humidified chamber for one hour at room temperature. Add 500 microliters of Ki-67 primary antibody and incubate overnight at four degrees Celsius. Wash the cells again three times in PBS for five minutes each.
Add one milliliter of the secondary antibody and incubate the cells for one hour at room temperature in the dark. After removing the secondary antibody solution, wash the cells again three times for five minutes in PBS in the dark. To label the HEI-OC1 cells with phalloidin, incubate them in 1:200 phalloidin for 30 minutes at room temperature.
To label the cell nuclei, incubate the cells with five micrograms per milliliter of DAPI For 10 minutes at room temperature. After delivery of the transgene mice, genotype the pups using a primer set specific to the CBRb1 fusion region as described in the manuscript. Breed adult TetO-DN-CB-myc6-Rb1 mice to the ROSA-CAG-rtTA and tetrecycline inducer line to generate experimental DN-CBRb mice, and genotype their offspring as described in the manuscript.
NIH3T3 does not express endogenous RB1 protein, so the robust RB1 expression seen here is due to transgene induction in the presence of Dox, but not in its absence, confirming the efficient activation of the TetO-DN-CB-myc6-Rb1 mix RB1 construct. In contrast, HEK293 cells have endogenous RB1 expression. The addition of doxycycline led to TetO-DN-CB-myc6-Rb1 activation and RB1 down-regulation.
RB1 expression was resumed 21 hours after doxycycline was removed from the media. HEI-OC1 cells cotransfected with the same vectors, displayed a modest but significant increase in cell number follow doxycycline treatment compared to transfected cells not treated with doxycycline, and the untransfected cells. Fluorescent in situ hybridization confirmed the genomic insertion of TetO-DN-CB-myc6-RB1 transgene in mice.
There was an initial increase in total RB1 protein expression in cochlea of the transgenic mice treated with doxycycline for three days compared to the control group. However, in groups treated with doxocycline for seven and 10 days, a significant reduction in RB1 expression was observed. Regardless of the tissue analyzed, a significant reduction in RB1 protein was observed in transgenic mice treated with doxocycline, but not in the control group, confirming efficiency of the TetO-DN-CB-myc6-RB1 construct.
While attempting this procedure, it's important to verify that each of the coding elements of the transgene are in frame with each other. If the coding regions of Cathepsin B and retinoblastoma described in this report were, by chance, connected out of frame, it wouldn't yield the intended fusion protein. Thus, DNA sequence verification must be done before injecting the transgene into single-celled mouse embryos.
Following this procedure, the ubiquitous CAG promoter can be replaced with specific promoters in order to study gene function in a cell's specific manner.
