HI,

did you check for HA tag or transgene expression itself? Are you sure about the reading frame?

Sven

Hello Dr Sven,

Yes, I have sequenced the whole frame and found it content. And the reading frame is absolute. Thanks.

Pratibha

Hi,

What are your controls?

Do you have other LVs with HA tag already validated in the past and which could be used as positive control of transduction and detection?

Or GFP vector?

This is a large protein that you want express, so this is not easy but generally possible, however, for success, you have to optimize all parameters: design of expression cassette, LV production (very high titer), LV transduction, detection. So this is important to have controls at each step to know which step(s) need to be optimized to have positive results.

You can have a look here about protocol of transduction:

https://www.geg-tech.com/knowledge/lentiviral-vectors-multifaceted-tools

Let me know if you have questions,

Best

Nicolas

Hi Nicolas,

I do have AVs as a positive control and even I include a negative control to the same set of experiment. All I have been succeed is getting positive control is expressing well but my construct has been failed to overexpress.

I guess my experiment is optimised but I am not sure about the construct. It has been sequenced well. Thanks!

Pratibha

Just to be sure, your vector of interest is a lentiviral vector?