3 replies to this topic

[jackster101]

Hi-

 

I'm confused! I have 2 vectors-

pRetroX-Tet-On Advanced   &

pRetroX-Tight-Pur (with my gene of interest)

 

My question: Tell me if I'm doing this right---I was planning on taking regular 293T cells and doing an infection into 293T (like I do for lenti)- with VSVg, delta, pRetroX-Tet-On Advanced vector using Fugene--harvest the virus and infect my cells, MCF-7 and generate a G418-resistant stable line that expresses the transactivator.

 

I was then going to do another infection into 293T like before but use pRetroX-Tight-Pur (with my gene of interest). And then infect the MCF-7/pRetroX-Tet-On Advanced line with sup that I generated---Does that sound right?

 

It seems odd that I'm using the same infection protocol for retro that I have used for lenti??? 

 

Are infection protocols similar. I remember from years ago--people would use Phoenix cells than 293T

[jackster101]

Anyone??

[CPRES]

293T is the parent cell line for Phoenix cells.

 

- With 293T, you will need additional viral packaging vectors (2) co-transfected along with your expression vector.

 

- With Phoenix cells, you get in-build packaging machinery (gag, pol, env genes), so you need to transfect the cells with only your expression vector.

[jackster101]

Thanks! Got it