The overall goal of this procedure is to demonstrate a novel strategy for site directed mutagenesis in anomalies mosquito cells. This is accomplished by first preparing healthy cultured cells for transfection. The cells are then transfected with the appropriate control and test DNAs through the addition of transfection reagents.
The next step of the procedure is to monitor the control cells for fluorescence and to reduce the density of the experimental cells. Finally, the gene conversion frequencies can be determined in the experimental cells using flow cytometry at five to seven days post transfection. Ultimately, results can be obtained that show site directed mutagenesis of an episomal gene target through locked nucleic acid oli nucleotide induced DNA repair.
Hi, I'm Dr.Nazy Pacor from the laboratory of Dr.Shirley Lockhart in the Department of Medical Microbiology and Immunology at the University of California Davis. Today we'll show you a novel procedure for the site directed mutagenesis of anomalies mosquito cells. We use this procedure to study the function of mosquito innate immune response genes through site directed mutagenesis.
So let's get started. Prior to starting the protocol, obtain mosquito cells that are healthy and approximately 80%confluent. If adherent, overgrown or unhealthy cells will result in low transfection efficiency and will give inconsistent results.
This protocol is demonstrated with ananais Gambier SUA five B cells. However, Ananais ensi MS Q4 three cells can also be used. Begin the transfection setup by warming S two media in a 28 degree Celsius water bath for 30 minutes.
The all transfection reagent materials, plasmid and oligonucleotides on ice. Using sterile technique in a tissue culture hood aspirate off the old media without disturbing the cells at an equivalent volume of fresh warmed media and rinse the cells off the bottom of the flask. Using a pipette, the concentration of cells should then be determined and adjusted to a final concentration of one times 10 of the six cells per milliliter.
Once the final concentration has been adjusted, add one milliliter of mosquito cells to each well of a six. While plate cells can be kept at room temperature while preparing the transfection reagent materials, trans transfer plasmid DNA to sterile micro centrifuge tubes. Here green fluorescent protein expressing plasmid is used as a positive control and blue fluorescent protein expressing plasmid is used as a negative control.
The test DNA also includes the BFP expressing plasmid, but with increasing concentrations of BFP specific locked nucleic acid, modified oligonucleotides or LNA os. After the DNA has been aliquot, add 99 microliters of kage and EC buffer for each microgram of plasma DNA and L-N-A-O-N. Then slowly add eight microliters of enhancer per one microgram of DNA used vortex, the DNA buffer enhancer mixture.
For one second, spin the solution down briefly and incubate the samples at room temperature For five minutes, add 10 microliters of affecting reagent per one microgram of plasma DNA and vortex For 10 seconds. Briefly spin the solution to the bottom of the tubes and then incubate at room temperature for 10 minutes. Following incubation, bring the final volume of each tube to 1000 microliters by slowly adding fresh warmed media to the DNA buffer enhancer effecting mixture DNA may precipitate at this point if the media is added too quickly, gently mixed by pipetting up and down three or four times.
Finally, slowly add one milliliter of the appropriate DNA enhancer affecting mixture to each well of cells. One drop at a time. Gently swirl the plate to ensure uniform distribution of cells and transection mixture.
Incubate the cells under normal growth conditions one day after transfection, gently aspirate the media off the cells and slowly replace it with an equivalent volume of fresh warmed media. Take care not to disturb the cell layers at the bottom of the wells. Two days after transfection, examine positive control cells transfected with GFP expressing plasmid under a fluorescent microscope to determine the transfection efficiency.
Four days after transfection s uua, five B cells must be thinned as they grow very quickly and will become overgrown. To do this, aspirate the old media off the cells and slowly add an equivalent volume of fresh warmed media. After adding new media to the wells, detach all the cells from the bottom of each well by pipetting up and down, discard half the volume of the cell mixture and add one milliliter of fresh warmed media to bring the final volume up to two milliliters.
Check the cells daily to make sure they're healthy and not overgrown. Thinning them as necessary. Allow the cells to grow under normal conditions for five to seven days post transfection before collecting them for analysis.
At five to seven days post transfection collect the cells by aspirating the old media off the cells and adding one milliliter of sterile room temperature PBS to each. Well detach the cells from the bottom of each well by pipetting up and down. Finally, transfer the cell mixture to five milliliter snap cap tubes and immediately analyze the cells via flow cytometry.
A defense eye. MS Q4 three and a gambier UA five B cells were transfected with positive control GFP expressing plasmid. The cells are shown under light and fluorescent microscopy and as emerged image flow cytometry data confirms the conversion of BFP to GFP via mutagenesis of a single nucleotide following transfection with BFP specific LNAO Ns.SUA five B cells were transfected with the GFP expressing plasmid as a positive control with A BFP expressing plasmid as a negative control or with BFP expressing plasmid and increasing concentrations of BFP specific LNAO ns.
The gating strategy for flow cytometry data shows forward versus side scatter of live cells, propidium iodide negative cells and GFP positive cells five days following transfection, the percentage of GFP positive cells following transfection with BFP expressing plasmid and increasing concentrations of LNAO NS can be seen here in graphical form confirming the conversion of BFP to GFP by L aos. We've just shown you a novel procedure for the site directed mutagenesis of anomalies, mosquito cells. When doing this procedure, it's important to remember to make sure that your cells are healthy and at the right density prior to transfection.
So that's it. Thanks for watching and good luck with your experiments.
