An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Our work generally focuses on using synthetic genetic devices to manipulate stem cell faith and behavior. By controlling cellular states, we aim to advance the development of next generation cellular therapies. Recently, a deeper understanding of the relationship between the dosage of a faith determining gene and the cells lineage has been established.

This was facilitated by the development of genetic controllers that are able to titrate a given gene precisely. Classic molecular biology techniques such as transfection or lentiviral transduction for simple gene over expression will always be a mainstay. However, our field has recently adopted synthetic devices that incorporate linear and non-linear feedback in order to obtain more complex outputs from delivered DNA.

Gene dosage has a key role in determining the outcome of gene expression. When delivering multiple exogenous genes, a key experimental factor is how much of each gene will give the optimal or desired outcome. Our protocol allows researchers to rapidly scan across dosages in a single treatment, looking for the desired ratio of DNA devices.

To prepare a gelatin coated 24 well plate add 200 microliters of these 0.2%gelatin solution to each well. Gently shake the plate to evenly spread the solution and allow the wells to coat at room temperature. After 15 minutes, using a vacuum aspirator, carefully remove any leftover gelatin from the wells.

Add 0.5 milliliters of freshly prepared NBIL media to each well and place the plate in a tissue culture incubator to prewarm. Aliquot 30 milliliters of wash media into a 50 milliliter conical tube. Aspirate the media from the tissue culture dish of MPSCs and add required amount of cell detachment medium.

After three to five minutes, pipette up and down 15 to 20 times using a P1000 pipette. Observe the cells under a microscope to ensure a single cell suspension. Transfer the cell suspension into a 50 milliliter tube containing wash media.

Centrifuge the suspension at 300G for five minutes at room temperature and aspirate the wash media. Resuspend the pellet in approximately 300 microliters of wash media. To begin, aliquot 200 microliters of NBIL media into 1.5 milliliter tubes.

Add 26 microliters of the optimum transfection reagent mixture to the DNA optimum mixture. Pipette the reagents vigorously, approximately 10 times. Transfer the required volume of MPSC suspension to the 200 microliters of NBIL tubes.

Then add lipofectamine 2000 and DNA mixture to the tubes and mix well by pipetting. After five minutes, centrifuge the mixer at 300G for five minutes and remove the supernatant, leaving approximately 50 microliters in the tube. Resuspend the pellet in 100 microliters of NBIL media.

Transfer the cell suspension to a gelatin coated 24 well plate and place it plate in the incubator. Transfer the required volume of MPSC suspension to seed 25, 000 cells per well of a gelatin coated 24 well plate and place the plate in the incubator. One hour prior to transfection, replace the media from the wells with 0.5 milliliters of NBIL media.

After incubation, dropwise add lipofectamine 2000 and DNA mixture to the wells. Place the plate in the incubator for 48 hours. After transfection of MPSCs, aspirate the media from the six well plate.

Then, add 200 microliters of tripson to each well, swirl, and incubate for 30 seconds at 37 degrees celsius. Tap the side of the plate and add 200 microliters of ice cold fax buffer. Using a P200 pipette, mix the contents of each well to dislodge cells and make single cell solutions.

Transfer 200 microliters from each well to the appropriate well on the 96 well plate. Centrifuge the plate at 300G for five minutes. After centrifugation, resuspend the pellets in 150 microliters of fax buffer before running the sample on a flow cytometer.

Compared to reverse transfections, forward transfections showed a significantly lower proportion of cells positive for the marker. Interestingly, forward transfections did not deliver a significantly lower amount of DNA on average to the population of cells. In reverse transfections, incubation time significantly influenced the percentage of reporter positive cells.

However, the average reporter expression in positive cells was not affected by incubation time. For both poly and co-transfection, a forward transfection significantly decreased the number of cells present at the chosen endpoint, compared to reverse transfection. In the case of reverse poly-transfections, higher transfection efficiency and a broader dynamic range of well-represented DNA ratios were observed.