This method can help answer key questions, such as how to positively moderate the activity of a gene, by taking advantage of mRNAs that are present in the cells and tissues. How to compensate for deficiencies in activity, and how to achieve high translation from mRNAs in vitro and in vivo. The main advantage of this technique is that we can globally screen SINEUP's target genes in living cells, by imaging without fixation and collection.
So the implications of this technique extend to our therapy of haploinsufficient instances, because SINEUPs can induce two-fold increase in a deficient protein, such as frataxin in cases of Friedrich's ataxia. To begin, open the Zenbu browser, and click on Fantom project database of interest. Search the target gene, and check for transcription start site, or TSS, and expression level in desired cells and tissues.
To determine TSS, consult the example analysis of Parkinson's Disease protein seven, or PARK7 mRNA. Check TSS by cage peak region. To determine cell and tissue specific transcription expression levels, select the cage peak region and click on magnify genomic region to selection.
Check PARK7 expression in the cell and tissue type of interest, by exploring the transcript expression list at the bottom of the page. Then, design binding domain sequences of several different lengths corresponding to 40 bases upstream, and 32 bases downstream of the first methionine or AUG. Before transfection with SINEUPs, seed cells of interest on two poly-d-lysine coated 24 well plates, as described in the manuscript.
Incubate for 24 hours at 37 degrees celsius in a five percent CO2 incubator to achieve 70 percent confluency. It is very important to distribute cells equally in the wells, for this purpose gently shake the plate 10 times back and forth after seeding the cells inside a clean bench, and repeat in the five percent CO2 incubator. After the incubation, change the medium to 0.4mL of fresh medium.
For analyzing SINEUP-GFP make several 1.5mL tubes with 0.1mL of mixed solution, with pEGFP-C2, SINEUP-GFP, transfection reagents and OptiMEM in each tube, and then incubate the tube at room temperature for 20 minutes. Add 0.1mL of this mixed solution from each tube to a separate well of a 24 well plate, and label these wells with SINEUP-GFP one, two, three, four, and five. Incubate the plate at 37 degrees Celsius in a five percent CO2 incubator for 24 hours.
After 24 hours, wash the cells with 0.5mL of PBS. Add 25uL of 0.05 percent weight per volume trypsin, and incubate in a five percent CO2 incubator for five minutes. Add 275uL of cell medium per well.
For protein extraction, take one of the two plates, and harvest 225uL or three quarters of the cells from one well, and 75uL or one quarter of the cells for RNA extraction, each into a separate 1.5mL tube. Centrifuge at 6000xg for five minutes at four degrees celsius. After centrifugation, carefully remove the supernatant from the tube labeled by protein isolation.
Add 60uL of lysis solution to the cells and mix by pipetting Mix thoroughly by rotating at slow speed for one hour at four degrees Celsius. Centrifuge the tube at 14, 000xg for 10 minutes at four degrees Celsius to collect protein supernatant. To determine protein concentration, first prepare five to six times dilution of fresh bovine serum albumin protein standard in ultra-pure water, with concentrations ranging from 0.2 to 1.5mg/mL protein.
To prepare working reagent A Dash mix add 20uL of reagent S to one mL of reagent A in a two mL tube. Add five uL of water as a negative control. And then, BSA standard or protein sample in each well.
Add 25uL of reagent A Dash, and then carefully add 200uL of reagent B per well, avoiding any bubble formation. Cover the plate with aluminum foil to incubate at room temperature, for five to eight minutes. Measure the protein absorbance at 750nm with a spectrophotometer.
Prepare a standard curve by plotting BSA standard protein concentrations on the x-axis, and their respective absorbance on the y-axis. Apply a standard curve equation to calculate the sample protein concentration. To start protein separation, add one volume of 2x loading dye to each volume of the protein sample.
Heat at 90 degrees Celsius for five minutes, and immediately cool on ice for one minute. Load 10 to 20ug of protein samples to 10 percent SDS polyacrylamide gel and separate at 100 to 150V. Transfer the protein from the gel, to a 0.5 micrometer nitrocellulose membrane, by a semi-dry transfer instrument.
Fill with transfer buffer at 25V for 30 minutes. After the transfer, place the membrane in a container, and add blocking solution until the membrane is completely soaked, and incubate it at room temperature for 30 minutes with shaking. Add the appropriate antibody to the blocking solution, and pour over the membrane.
Hybridize by incubating the membrane for 30 minutes at room temperature, while shaking. After hybridization, wash the membrane by adding 1x TBST buffer for five minutes at room temperature, and repeat two more times. Perform the next hybridization with a secondary antibody diluted in blocking solution over the membrane, at room temperature for 30 minutes while shaking.
Then wash the membrane by adding 1x TBST buffer for five minutes at room temperature, and repeat two more times. Transfer the membrane to a box filled with two mL of HRP enhanced ECL reagent mix. Cover the box with aluminum foil, and incubate for one to two minutes at room temperature.
Carefully remove the membrane from the ECL reagent mix, and expose it using a luminescence imaging instrument. To analyze the effect of SINEUP-GFP in cells by imaging, wash the pEGFP-C2, and SINEUP-GFP transected cells from the second 24 well plate with 0.5mL of PBS per well. To stain the nucleus, add 2ug of Hoechst-33342 to each well, and incubate cells at 37 degrees Celsius, for 20 minutes.
Use a high throughput microwell image cytometer to measure Hoechst stained cells, to count the total number of cells. Measure the intensity of green fluorescence to count the number of GFP positive cells. To analyze, protein upregulatory effect of the SINEUPs, use imaging software to determine a GFP integrated intensity Calculate it as the sum of all pixel intensities displaying signals in segmented objects obtained for each channel.
After a successful transfection with SINEUP-GFP, a synthetic SINEUP containing both an optimum binding domain and an effector domain GFP, mRNA translation was upregulated without changing the expression of GFP mRNA. A protocol of semi-automated image analysis improved detection time and increased the number of samples being simultaneously screened compared with a conventional Western blot analysis. Although there was a difference in signal intensity, from 2.6-fold in Western blot analysis to 1.4-fold in imaging analysis, significantly higher levels of GFP fluorescence were detected compared to the control.
While attempting this procedure, it's important to remember to identify the correct sequence of target mRNAs to design the correct binding domain. Genes are often transcribed from promoters, and may use different translation initiation site. The Zenbu browser is a very useful tool to explore such mRNA variance.
We suggest also design different SINEUPs with variable binding sites. Altogether we believe that quickly adapting SINEUPs to a multiplicity of targets will establish a new field consisting in positively regulated translation of existing RNAs, which is a reciprocal and complimentary to siRNA's field. A part of those function we envisage that this technology will be instrumental to produce larger amounts of proteins in vitro, like bioreactors, as well as in vivo to naturally correct the gene dosage.
