Hi. I have done a gene knockdown in a cancer cell line and it can be readily observed that the knockdown cells detach much readily during passaging by trypsin. Assuming that it indicate loss of cell adhesion, can I anyhow measure it? and what can be possible explanations?
hi Mohit Arora
If you are finding the significant trypsinization timing difference, sometimes it could happen because of different trypsin production batches, the temperature of enzyme activation, and high cell passage numbers otherwise it may be an arbitrary thing. Since, you are discussing cancer cells then you can analyze the cell adhesion molecule's expression after gene knockdown especially Ncad & Ecad, integrins etc. Even you can perform some protease enzyme activity assays to understand the reasons behind it.
Regards
Saurabh
Hi Mohit Arora ,
Crucially, what gene did you KO? If the effect on cell adhesion is real, ie not due to a different trypsin batch or temperature issues as pointed out by Saurabh Mandal , then my guess would be that this is a membrane protein, or something involved in cytoskeleton structure. The first thing to do is to look up the function of the gene, its localisation and interaction partners, that will give you a clue or many clues as to what might be going on. Or was it a transcription factor that regulates expression of membrane proteins? without knowing what the gene is, it is very difficult to give an explanation.
Cheers
Alena
Anything that affects the cell metabolism would affect adhesion even the protein you knocked out has nothing to do with cell adhesion.
Thank everyone for their answers. I would like to add that protein is a peptidase THOP1. It works at the terminal stages of protein turnover. However, its role in cell adhesion has not been reported yet. Further suggestions are welcomed. Saurabh Mandal Alena Pance Bill Chi Shun Ho
I observed a similar change in my HEK293 edited cells. In addition, the doubling time of my cells reduced drastically.
One reason might be that the transfected cells are stressed. Anyhow, you can measure adhesion by doing adhesion assay with or w/o ECM coating
Hi Mohit
There is one simple explanation for this observation. It’s possible gene you have knockdown encode for a protein, which is inhibitor of trypsin. That might be the reason! Best of luck! 🤞
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