Hi,

First of all I would check the construct. You can use some programs like Ape to see if your region of interest would be translated in frame with your current construct. Also check all components like poly A signal, promoter, etc if they are not accidentally being cut.

Another reason could be transfection problem. For this I would suggest you to include a positive control plasmid that you know worked before. This will tell you if your transfection method is fine or not. I won't expect any compatibility issue since HEK293 is well established transfectable cell line with high efficiency. I would rather check the method and reagents.

For western my advise also include a positive control sample, or at least an internal control like actin or tubulin just to be sure that everything is fine with your western blot.

I'm not sure why you need to wait 1 month to check the transfection. I think 2-3 days should be sufficient to check if your transfection is working or not.

Good luck!

Hi,

First of all I would check the construct. You can use some programs like Ape to see if your region of interest would be translated in frame with your current construct. Also check all components like poly A signal, promoter, etc if they are not accidentally being cut.

Another reason could be transfection problem. For this I would suggest you to include a positive control plasmid that you know worked before. This will tell you if your transfection method is fine or not. I won't expect any compatibility issue since HEK293 is well established transfectable cell line with high efficiency. I would rather check the method and reagents.

For western my advise also include a positive control sample, or at least an internal control like actin or tubulin just to be sure that everything is fine with your western blot.

I'm not sure why you need to wait 1 month to check the transfection. I think 2-3 days should be sufficient to check if your transfection is working or not.

Good luck!

Hi,

the suggestion of Raymond is really good and you should check the expression of your target after 24 or 48 hours after transfection. on the other hand, you have to be sure that your cells are not HEK293T cells since they contain a T antigen and  are resistant to G418. for this reason, G418 can not be used to generate stable cell line with HEK293T cells.

good luck!

Was your plasmid linearized? Anyway genome integration via transient transfection is very inefficient. You need to use a viral transduction instead.

Consider using a Lonza nucleofector because it is extremely efficient (expensive though). We've had very good success with that approach. Regarding the incubation period: I think you need to wait considerably longer than 24-48 hrs after transfection to check for expression. You will see a mix of transiently and stably expressed clones. Our approach (in establishing stable Nav channel lines with HEK cells) utilizes 600 ug/ml G418 (10-cm dish), 10-day wait period (with intermittent removal of dead cells). Then continued growth, until small colonies become visible. Move the individual clones over to a 12-well dish and keep maintaining them. Conduct functional testing when you're ready to expand from 12-well to T25. That may well be 4 weeks after the initial transfection. 

Make sure you are not using HEK293T cells (immortalized with large T and neomycin resistance)...could explain your problem since they are resistant to G418/neomycin. Just test your untransfected cells for resistance/sensitivity to confirm.

You should also test acute (1 or 2 days) transfections by Western, to confirm proper expression of your construct.

There may be more complicated explanations but the 2 above issues (resistant cells, bad constructs) are more likely.

Good luck!

I too agree with Raymond.