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question about Clonetech Retro-X Tet-on system

inducible system

Best Answer CPRES, 13 January 2015 - 12:46 PM

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.

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#1 jackster101

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Posted 12 January 2015 - 04:42 PM

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 VSVgdeltapRetroX-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



#2 jackster101

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Posted 13 January 2015 - 06:56 AM

Anyone??

#3 CPRES

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Posted 13 January 2015 - 12:46 PM   Best Answer

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.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#4 jackster101

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Posted 14 January 2015 - 08:36 AM

Thanks! Got it





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