新たに単離したヒト絨毛細胞栄養芽層に対するsiRNAトランスフェクションおよびEMSA分析

The overall goal of this protocol is to demonstrate optimal conditions for the transfection of freshly isolated primary villous cytotrophoblasts and to present a method for the identification of DNA protein interaction at promoter sites. This method can help answer key questions in the field of placenta research, such as those related to genes involves in cytotrophoblast differentiation and the regulatory mechanisms involved in their expression. The main advantage of this technique is that they're easy to perform and very efficient.

Furthermore, the time needed to conduct this experiment is short and can include a lot of different samples. Demonstrating these procedures will be Chirine Toufaily a former Phd student from my lab, as well as Yong Xiao, a research associate. Here, primary villous cytotrophoblasts are isolated from fresh placentas according to standard protocols.

The first step of this protocol is to analyze the purity of the preparation by FACS analysis. First, spin one times ten to the sixth of the freshly isolated cytotrophoblasts in micro centrifuge tubes at 300 times G for five minutes. Following the centrifugation, discard the supernatant.

Add one milliliter of pre-warmed PBS and repeat the centrifugation. After removing the PBS by aspiration add one milliliter of cold methanol to the cells and incubate at minus 20 degrees Celsius for 20 minutes. Next, after pelleting the cells by centrifugation, remove the methanol by aspiration.

Then add one milliliter of room-temperature PBS to the pellet to rehydrate the cells. After spinning the cells and removing the PBS, add PBS containing FBS with FC receptor blocking reagent and incubate for 45 minutes at room-temperature to eliminate non-specific binding. Once the incubation time has elapsed, wash the cells twice with 500 microliters of room-temperature PBS centrifuging at 300 times G for five minutes each time.

After removing the last PBS wash, resuspend the cells in 100 microliters of room-temperature PBS. Then incubate the cells with a mouse monoclonal FITC conjugated anti-human cytokeratin 7 antibody clone LP5K, or a control isotype-matched non-specific antibody for 4 minutes at room-temperature in the dark. After washing the cells in PBS, analyze florescents by flow cytometry.

Use cell preparations that demonstrate a minimum of 96%CK7 positive cells by flow cytometry for further experiments. To transfect human primary villous cytotrophoblasts using a microporation device first prepare six well plates containing two milliliters of supplemented DMEM medium and place them in a 37 degree Celsius tissue culture incubator. Next, transfer aliquots of 1.5 times 10 to the 6th isolated primary cytotrophoblasts to micro centrifuge tubes.

Following a centrifugation to pellet the cells wash with one milliliter of Dulbecco's PBS. After removing the PBS, and gently resuspending the washed cells in 100 microliters of resuspension buffer add 300 nanograms of Syncytin-2 siRNA or controlled scrambled siRNA. And then aspirate the cell siRNA mixture using a 100 microliter transfection pipette.

Now insert the pipette into the transfection station and subject the cells to a single 1300 volt pulse of 30 milliseconds duration. Then immediately transfer the cells to the previously prepared six well plates and gently rock the plate for 30 seconds to ensure an even distribution of the cells. It's very important no to make air bubble while resuspending the cells and to transfer them immediately to the medium.

Otherwise this will affect that viability of cells and the transfection efficiency. Incubate the plate at 37 degrees Celsius in a humidified carbon dioxide incubator for 24 to 48 hours. First, wash the cultured cytotrophoblasts twice with one milliliter of pre-warmed DPBS.

After aspirating the DPBS add 500 microliters of 0.25%Trypsin-EDTA and incubate for one to three minutes at 37 degrees Celsius in a carbon dioxide incubator. Next, stop the Trypsin treatment by adding 500 microliters of serum containing growth medium and transfer the cells from each well into a 1.5 milliliter micro centrifuge tube. Pellet cells by centrifugation at 300 times G for two minutes at room temperature.

After washing and pelleting the cells once more, carefully remove all supernatant without disrupting the pellet and place the tubes on ice until protein extraction. Prepare the phosphorylation reaction in a micro centrifuge tube by adding 15 nanograms of a forward oligonucleotide along with 1XT4 Polynucleotide kinase buffer, one unit of T4 Polynucleotide kinase, and 20 microcuries of gamma-32P labeled ATP to nuclease free water, for a final volume of 20 microliters. Incubate the reaction at 37 degrees Celsius for 30 minutes.

Then, stop the reaction by adding 5 microliters of 0.5 molar EDTA. Bring the volume up to 50 microliters with nuclease free water. Then proceed to the removal of unincorporated nucleotides and the hybridization of the complimentary strand according to the instructions in the written portion of the protocol.

Setup the DNA binding reaction by first adding 10 micrograms of nuclear extract 1X gel shift binding buffer to nuclease free water for a final volume of 10 microliters. For the competition reaction add one microliter of cold non-specific or specific double-stranded oligonucleotide. Next, add one microliter of the radioactive oligonucleotide probe to each reaction.

Incubate the reaction at room-temperature for 20 minutes. Then add one microliter of 10X gel loading buffer per reaction. Load each sample onto a non-denaturing polyacrylamide gel and run the gel for 1.5 to 2 hours at 150 volts.

After migration, wrap the gel and the glass in plastic wrap. Then position the wrapped gel in an exposure cassette and place a phosphorus screen over it. Leave the cassette at 4 degrees Celsius for 24 hours.

The next day remove the screen from the exposure cassette and insert it into a phosphorimaging device for scanning. Microporation of a scrambled control or a Syncytin-2 specific siRNA in villous cytotrophoblasts is compared to a lipid based transfection approach by western blot detection of Syncytin-2 and the control protein GAPDH. Syncytin-2 protein levels were normalized in terms of band intensity with corresponding GAPDH signals.

Microporation conclusively leads to a more significant knockdown of Syncytin-2 expression. This image shows an EMSA analysis of nuclear extracts villous cytotrophoblasts incubated with a probe corresponding to a promoter region of Syncytin-2 with or without an excess of specific, non-specific a cold oligonucleotide for 24 or 48 hours. The Syncytin-2 promoter drived probe showed the presence of two DNA protein complexes.

Addition of cold wildtype oligonucleotide competed for the formation of the complex while no competition was observed with a cold, unrelated, oligonucleotide. While attempting this procedure, it's very important to carefully handle cytotrophoblasts after transfection and to transfer them immediately to the medium. Following the EMSA procedure, a supershift assay can be performed in order to identify transcription factor that bound to DNA.

After watching this video you should have a good understanding on how to handle and carefully transfect a cytotrophoblast as well as how to perform an EMSA assay with these kind of cells.