So for the last couple of decades, the importance of microRNA has been recognized in human disease conditions, but the problem is that, therapy-wise, it did not go to the primetime. One of the problem is, one microRNA can have different targets in different tissues. So one microRNA can be protected in one organ but detrimental to the others.
So this method can help to eliminate tissue-specific microRNA knockdown strategy. The main advantage of this technology is we will use a microRNA sponge delivery and can be performed a tissue-specific manner. Begin this procedure with sponge design as described in the text protocol.
To remove the CMV promoter from the commercially-available EGFR plasmid, first perform a digestion reaction. Following digestion, add five times the reaction volume of column binding buffer. Purify the digested linearized plasmid with a DNA purification column as described by the manufacturer.
Elute with 25 microliters of elution buffer added carefully to the center column. Gel-purify the digested plasmid on a 0.5%agarose gel by first adding five microliters of loading buffer to the eluded plasmid. Load the entire sample into one well on a 0.5%agarose gel.
Include a separate lane with 500 nanograms of uncut vector. Run it for 30 to 40 minutes, until an adequate separation of cut and uncut plasmids is achieved. After cloning the cardio-specific promoter alpha-myosin heavy chain as subscribed in the text protocol, add five times the reaction volume of the column binding buffer to the PCR reaction and purify it with a DNA purification column as described by the manufacturer.
Elute the mixture with 25 microliters of elution buffer added carefully to the center of the column. Add five microliters of loading buffer to the eluded plasmid and load the entire sample into one well of a 1%agarose gel. Run the gel for 30 to 40 minutes before excising the PCR product as before.
Ligate the alpha-myocin heavy chain promoter with the EGFP plasmid at 50 degrees Celsius for 15 minutes before cooling the reaction on ice. Perform a bacterial transformation by transfecting 50 microliters of competent cells with 2.5 microliters of infusion ligation mix. Rest the Eppendorf tube on ice for 20 minutes.
Then, incubate the reaction in a 42 degree Celsius water bath for 40 seconds and place the tube on ice for two minutes. Following the transformation, add 350 microliters of SOC media and grow the cells at 37 degrees Celsius for 45 minutes. Plate 200 microliters of the outgrowth solution on LB plates with Kanamycin.
Perform a colony PCR to identify Kanamycin-resistant cells, which contain the alpha-myosin heavy chain promoter ligation. Ligate the microRNA 181 sponge into the alpha-myosin heavy chain expressed EGFP vector by setting up a 21 microliter ligation reaction as detailed in the text protocol. Perform the ligation at room temperature for five minutes and then place it on ice.
Transform 50 microliters of XL1-Blue competent cells with two microliters of ligation mix as before. Perform a colony PCR to identify Kanamycin-resistant bacteria, which contain the correct plasmid cloned with the first alpha-myacin heavy chain followed by the microRNA 181 sponge construct sequence. Design screening primers to PCR across the ligation junction.
Expand the PCR positive clones in LB Kanamycin broth and in plasmids purified using standard mini-prep plasmid purification protocols. Perform DNA sequencing to verify that the plasmid is expressing the desired DNA sequences. Using an electroporator, transfect rat myoblasts with either a scramble sponge as a control, or the plasmid cloned with the first alpha-myocin heavy chain, followed by the microRNA 181 sponge construct.
Briefly, transfect 0.4 million cells with two microgams of plasmid DNA and 100 microliters of a solution compatible with the electroporation device. Use an appropriate program to transfect the H9C2 cells. Plate only the GFP-expressed cells into a six-well plate with an addition of neomycin to the complete growth media.
After seeding five to 10 cells in each well of a 96-well plate, followed by flow sorting, expand the monoclonal cells from the 96-well plate to 24-well plates over several passages. Prepare the liposomal nanoparticles by dissolving the components listed in the text protocol in a mixture of chloroform and methanol in a glass vial. Prepare the mixture by adding 5%glucose to the vacuum-dried lipid film before leaving it overnight to allow this mixture to hydrate the film.
Gently rock the vial for two to three minutes at room temperature in order to produce multilamellar vesicles. Then, incubate the vesicles with occasional shaking in a 45 degree Celsius incubator. Sonicate the multilamellar vesicles to prepare small unilamellar vesicles in an ice bath for three to four minutes, until clarity can be observed.
Add a 100%duty cycle and 25 watts of output power. Mix either a scramble sequence or a microRNA 181 sponge sequence cloned into the alpha-myocin heavy chain expressed EGFP plasmid with liposome on a one-to-three charge ratio basis to form the nanovector. The results of optimized three-week treatment protocols with six intravenous injections through the tail vein are shown here.
The yellow colorization in the epifluorescence image demonstrates expression of the EGFP vector. The maximum intensity of yellow colorization is observed at the week three time point. The alpha-myosin heavy chain promoter sequence in the EGFP vector selectively expresses either microRNA 181 sponge or scrambled sequence in the heart at Day 21 as revealed by the yellow fluorescence.
The Western blot and band densitometry showed a significant increase in the mt-COX1 expression in the microRNA 181 sponge nanovector injected rats as compared to the scramble nanovector groups, with alpha-Tubulin as the normalization control. One of the major challenges in this particular protocol is to keep the orientation of the sequencing, you need directional. Once a bacterial preparation is made, with a proper orientation of the microRNA sponge, this technique can be done with a two or three minute tail vein injection time.
While attempting this procedure, it is important to sequence your bacterial colony multiple times, multiple batches, to make sure the microRNA response sequence is in the correct order. After watching this video, you should have a good understanding of how to design a microRNA sponge to knock down the expression of any microRNA family and prepare an in vivo application of microRNA sponge. Following this procedure, other methods, such as in vivo antagomir treatment can be performed to answer additional questions like whether a specific microRNA can be knocked down in all the organ at the same time.
After its development, this particular technology paved the way for researcher in the field of microRNA to explore tissue-specific knockdown of a microRNA in the pathophysiology of multiple-disease conditions. Tissue-specific microRNA inhibition is currently an undeveloped technique in the microRNA field. The importance of mat-down regulation of functional inhibition of up-regulated microRNAs in human disease suggest exploring microRNA biology to create a cellular vulnerability for therapeutic intervention.
