The overall goal of the following experiment is to quickly determine the role of the gene of interest in the maintenance of mouse embryonic stem cell or ES cell self-renewal. This is achieved by first assembling the transfection master mix that will be used to silence the genes of interest. As a second step.
The transfection master mix is transferred to a gelatin coated plate and T 4G IP cells are added. Cells are then cultured in normal ES cell culture conditions allowing the effect of the gene silencing to manifest After four days of culture, the cells are detached from the plate and their self-renewal status is determined by flow cytometry. Ultimately differences in the percentage of GFP negative cells between the control and experimental siRNA transfected T 4G IP cells can be evaluated by facts.
The main advantage of this technique over existing methods such as cell morphology or lineage marker analysis is that it provides higher sensitivity and throughput and is therefore better suited for medium to large scale genetic screens or genetic epistasis analysis. This method can greatly facilitate the study of the gene important of ESL maintenance and self-renew. It also can be adapted to the self specification events that occur during ESL differentiation.
T 4G IP cells are maintained in gelatin coated plates. They are plated at about two times 10 to the fourth centimeter squared and passaged every two to three days for transfection. In 96 well flat bottomed plates begin by coding the plate with 0.1%gelatin, then set up the master mix of Optum and Lipectomy 2000 and aliquot into a 96 well U bottom plate.
Incubate the plate for five minutes at room temperature, then add five picomoles I a to the lipid optimum mixture and incubate for another 15 minutes. Next, remove gelatin from the 96 well flat bottom plate and use a multichannel pipette to transfer the i a lipid complexes in optimum from the U bottom plate to the flat bottom plates. Now collect the OC 4G IP cells and resuspend them in fresh M 15, medium to nine times 10 of the fourth cells per milliliter.
Then use a multi-channel pipette to add 100 microliters of the cell suspension to each well of a 96 Well flat bottom plate pipette up and down three to five times to mix the cell suspension with the pre Eloqua. Sir, a lipids complexes changing the tips for different sir a transfect then place the plates in a 37 degree Celsius 5%carbon dioxide incubator and culture the cells for four days on each day of culture. Use a multi-channel aspirator to remove the old medium, being careful not to scratch the cells at the bottom of the plates.
Then use a multi-channel pipette to feed the cells with 100 microliters of fresh ES cell medium four days after transfection. Remove the medium using a multi-channel aspirator and rinse the cells once with 100 microliters of PBS. Then use a multi-channel pipette to add 25 microliters of 0.25%tripsin to each.
Well incubate the cells for five minutes at room temperature with occasional agitation. Then visually inspect the plate to ensure the complete detachment of the cells after the cells have detached. Add 90 microliters of PBS with 10%FBS to inactivate the trypsin, pipette the cell solution up and down several times to dissociate cells into a single cell suspension.
Finally, use the high throughput mode on the HTS unit of a fax analyzer to analyze 10 microliters of the cell suspension. Set the count threshold to 1.0 times 10 to the fourth cells and adjust the PMT voltage to capture the cells in the forward versus side scatter plot. Start the analysis gate for the live cell population in the forward versus side scatterplot and then create a histogram plot for the GFP channel.
Setting the gate in the GFP channel so that about 10%of the cells appear to be GFP negative in the mock or control SINA Transfected cells determine the percentage of differentiated cells based on the percentage of GFP negative cells. For each treatment. Compare the percentage of differentiated cells between experimental and controls irna transfect.
This first figure shows that wild type E 14 TG two A cells represented by the black histogram are negative for GFP while the T 4G IP cells represented by the green histogram are GFP positive when maintained as ES cells in these next two figures. Forward versus side scatter plots and histograms of the GFP channel of T 4G IP cells four days after transfection with control or T four siRNAs are shown in the scatter plots. Live cells are first gated in the forward versus side scatter plot as the dead cells and debris are GFP negative and increased background as seen in the histogram data in the controls irna transfected cells, the vast majority of the cells are GFP positive.
These bar graphs show the percent of differentiated cells from control nano OCT four and SOX two, siRNA transfected, OCT 4G IP cells four days after transfection. In all cases as expected, sir, a transfected cells were determined to exhibit higher levels of differentiation than controls. Once mastered, this technique can be performed one hour of hands-on time for each 96 well plate.
After watching this video, you should have a good understanding of how to use the Oxford GIP reporter assay to quickly determine the role of your gene of interest in mouse ESL maintenance.
