InI m working with htert rpe -1 cell line for cilia visualization. I want to see whether my gene of interest is affecting cilia morphology. My GOI (693bp) is cloned in p3x vector ( 6299bp ) which makes my plasmid almost 7kb. I m using lipofectamine 3000 for transfection. Whenever I transfection I feel like the cell is being stressed. Kindly suggest me a proper technique for better transfection efficiency and healthy visualization of these cells.
Hi Geetha,
I know you chose the hTERT-RPE-1 cell line to visualize cilia for obvious reasons. I also think this problem is not that much during siRNA transfection. This problem of transfecting plasmids, regradless of its size, is always there with the cell line. I also faced similar issues even while transfecting empty-GFP vectors. The exact reasons I’m not aware of, but possibly due its endogenous modification to make this terminally differentiated cell a propagative one.
What you can do to troubleshoot the condition is to use a very good quality RPE-1 cells and check transfecting some empty vectors first. Even before that you can keep your cells with only lipofectamine-3000 (without any plasmid or siRNA) to check whether the reagent that you're using is free of contamination and not causing the stress. Try to maintain the cell quality, else even after successful transfection you'll not be able to observe cilia (too much stress hinders with cilia formation in the cells).
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