We have been using a pMX based vector for retrovirus preparation (PlatE cells, Lipofectamine 3000 kit). This polycistronic vector was sent to us from another group. However, when we use the retrovirus that has been made with this polycistronic vector to infect fibroblasts (mouse), we are not seeing an effect and what is expected. Also, the results are not consistent.
On the other hand, when we do pMX dsRED as a negative control it works very well which implies there isn’t any problem with our retrovirus preparation protocol and reagents we use- PlatE cells, Lipofectamine 3000 kit.
I’m thinking the vector could be unstable as it is a polycistronic construct (size: about 13 kb). Is this possible? What can be done at this point to troubleshoot? May be design primers for this vector and send it for sequencing? Another way probably to test would be to transfect 293T cells with this polycistronic vector, isolate RNA from these cells and check for the expression levels of my gene of interest to make sure we have good transfection efficiency. I'm thinking of doing this too. Although, I’m not sure if I’m right. What’s a best and efficient way to troubleshoot this problem?
Your response is greatly appreciated.
Thank you!
Hi Joy,
Thank you very much for your suggestions. I really like the "reverse transduction" concept. Will try it next time.
Thank you your time.
~ Pratap
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