The overall aim of this procedure is to generate stable human cell lines with tetracycline inducible, S-H-R-N-A or CDNA expression. First transfect target cells with the tatar expression plasmid and select for blaster side in resistance. Then pick individual colonies and scream for cells that stably express the Tet repressor protein.
Next, transfect the best teta expressing clone with plasmids designed to modulate target gene expression under control of the Tet response element culture. The transfected cells under double selection then pick single colonies and screen for the desired Teton clones by western blotting. Ultimately, the resulting Teton clones can be used to examine the impact of specific targets on select biological parameters.
Demonstrating the procedure will be a postoc in laboratory. Marty Gomez Martinez Generate a vector that drives expression of the Tet Repressor and the Blain resistance gene from the same CMV promoter in two 10 centimeter tissue culture plates seed one time center, the six cells in standard growth medium, one plate for transfection with a tatar expression plasmid, and the other as a negative control for Blain selection. The next day transfect one plate with two micrograms of TTA expression plasmid using FU gene six.
Following the manufacturer's instructions, then incubate the culture overnight next to both plates. Add standard growth medium containing the optimal concentration of blastin for the experimental cell line being used after 24 hours, harvest the cells then label 10 centimeter plates for serial dilution and proceed to plate duplicates of serial dilution using standard growth medium containing five micrograms per milliliter blaster side in make sure to properly label plates containing the unresected or the transfected cells. Monitor the cells, specifically noting that all cells on the unresected plates have died within the first week.
Change the selection media every two to three days after one to two weeks when the blaster soin resistant colonies appear. Select plates that contain between five to 50 colonies to ensure that single colonies can be picked when the colonies have reached a diameter of approximately five millimeters. Isolate at least 12 independent clones using cloning cylinders to pick single colonies.
Transfer each clone into a separate well of a 24 well plate culture cells to confluence. Harvest and transfer the cells from each colony into individual wells of a six well tissue culture plate. Continue to culture the clones in selection media when cultures are cofluent.
Split the cells from each well into two single wells of a six well tissue culture plate For each clone to be tested, freeze one well of the confluence cells. Harvest the cells from the second well. For evaluation of tatar protein expression.
Perform a western blot on the clones using a lysate of the parental cell line as a negative control to detect the tatar protein. Use the mouse monoclonal anti Tet zero two antibody. Proceed to expand the three clones of highest testar expression.
Free stocks of each promising clone as soon as possible. Now the clonal cells are ready for examination of your molecular and cellular parameters of interest. By comparing the parental cell lines with the tatar expressing clones, Select the best clone with the highest tatar expression that does not display any alterations in the molecular and cell biological parameters of interest.
Generate PTER plasmids with HRNA inserts of interest. Also construct a PT REX desk 30 vector containing the CDNA of interest using gateway technology. Transiently transfect the parental target cell line with PTER or P T-Rex S 30 plasmids.
Using the transfection reagents of choice. Then harvest the transfected cells and evaluate target gene expression by immuno blossoms. On day one, see two plates of the best tatar expressing clone using standard growth medium supplemented with certified tetracycline free serum culture overnight.
The next day, transfect only one plate with two micrograms of A-S-H-R-N-A or CDNA expression plasmid using FU gene six. On day three, add standard growth medium containing either five micrograms per milliliter, blastin, and 500 micrograms per milliliter, eosin or blastin and G four 18. Then on day four, split the cells to create duplicate plates of dilutions using standard growth medium for double selection.
Check the cells daily for cell death response to antibiotic treatment and replenish the media every three to four days. After two to three weeks. When blaster aside in zein or blaster aside in G four 18 double resistant colonies begin to appear.
Select the plates containing five to 50 colonies to pick single colonies culture until colonies have reached a diameter of approximately five millimeters or bigger. With cloning cylinders, pick and transfer single colonies into separate wells of a 24 well tissue culture plate culture. The clones in medium containing maintenance concentrations of five micrograms per milliliter, blaster, soin, and 50 micrograms per milliliter.
Eosin or blaster soin and G four 18. When confluent split the cells from each well into one single well of a six well tissue culture plate. When confluent split the cells into three wells of a six well plate to culture to 100%confluence.
Freeze the cells from one well to test for test inducible expression, add one microgram per milliliter tetracycline to one well while leaving the second well of cells. Tetracycline free incubate for 24 to 96 hours using the shorter times for screening of induction of CD NA expression and longer times to determine S-H-R-N-A mediated depletion of endogenous proteins. Then harvest the cells and process for immuno blossoming using the appropriate antibodies.
Select at least two clones with the desired depletion or overexpression and expand each culture. Remember to free stocks of each promising clone as soon as possible. This initial characterization of a human retinal epithelial cell line for stable expression of tatar shows all RPE one clones expressing varying levels of tatar.
As expected, the parental RPE one cell line does not express the exogenous tatar protein. The variation in Tet R expression among the RPE one Teton cell clones reflects the plasmid integration sites. This experiment characterizes stable RPE one Teton cell clones that express RNAs directed against the MST three kinase three cell clones were chosen for further analysis.
After watching this video, you have a good understanding of how to generate stable human cell lines with that unregulated expression of your research RNA or CDNA of interest.
