I am planning to overexpress a transcription factor in INS1 cells and at the same time I like to knockdown another receptor protein in the same cells that is over expressed of transcription factor (let say transcription factor X). For over expression lipofectamine LTX plus and for knockdown lipofectamine RNAiMAX will be used. my protocol will be : First transcription factor X overexpression will be performed and after 4 hours media will be replaced with complete media and knockdown of the 2nd gene will be done and incubate cells for 48h. Do you think this procedure will work? constructive criticism and suggestions are welcome
Make a stable knocked out INS1 cell line of the first transcription factor using CRISPER or shRNA. After having stable knocked out INS1 cells, overexpress the second transcription factor. Or you can do vice versa make stable overexpression of the first TF and then knockdown/ out the second TF.
If you do not want to make stable cell lines, then transient transfection is also possible but you have to check the timing of transfections. let me know.
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