Does it mean that the cells have lost hERG gene expression?
Does cell confluency and cell density while freezing affect the cell line characteristics?
What is the role of selection antibiotic geneticin? Is there a possibility of cells becoming resistant to geneticin?
Have you verified KCNH2 expression by western blot or qPCR? Alternatively, you could check if the patch clamp yields a different result using cells from a low passage or initial preservation that should be optimally expression your GOI.
Normally, your 293 cells should be sensitive to G418. Beware that 293T cells are resistant because they carry a Neomycin resistance gene. While unlikely, you cells might have dropped the expression of your GOI and kept the expression of your resistance marker. Have you tested your G418 on naive cells?
Best,
Nicolas
I used cells from low passage and from different batches. But to my surprise the low current issue persisted still. Now I look towards the integrity of the instrumental protocol. The reason is that all the cells are giving very low current irrespective of the passage number and stable seals.
Hi Jaison,
What you are experiencing is actually not that uncommon... Other than issues with G418, passages, etc... that you have apparently excluded already, it is a possibility that expression of your channel is silenced (by methylation, etc...). It is quite common, especially with promoters like CMV. If it is the case then there is not much you can do other than developing a new line using a different vector/promoter.
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