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293t transfected with prtTA inducible vex


Best Answer jerryshelly1, 28 March 2014 - 10:50 AM

Sounds good. Can I get a vector name to satisfy my curiosity?

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#1 jackster101

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Posted 28 March 2014 - 05:12 AM

Hi-
I transfected a tet inducible, GFP tagged lentiviral vec into 293t to collect sup for infection of some cell lines. After 24 hrs I see intense GFP expression in 293t, but realized that I hadn't added tet yet! Is the vector leaky? What's going on??? Btw I didn't use tet free serum or tet free medium.,

Thx

#2 jerryshelly1

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Posted 28 March 2014 - 08:30 AM

Yes it sounds like you have basal expression of your GFP in the absence of Tet. This is not entirely uncommon. Maybe you should consider using a different Tet system to avoid abberant expression of your vector.



#3 jackster101

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Posted 28 March 2014 - 08:55 AM

I was just told that only my insert will be tet-induced with this construct--GFP is not inducible. I'll be ably to sort out my GFP positive cells (ones that took in the construct) without inducing with tet. 



#4 jerryshelly1

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Posted 28 March 2014 - 09:23 AM

What lentiviral vector are you using? Have you consulted the vector literature to see if your problem is included in the troubleshooting section?

 

Explain how GFP is not inducible.

 

Edit - I just reread my post and it sounds confusing. Yes, you confirmed your insert was present in your vector via sequencing and propagated that plasmid. You transfected this purified plasmid in your 293T cells, so there should only be your GFP tagged construct. I am confused to how you are going to sort out your cells. The presence of the GFP will indicate that your GFP tagged construct is 'leaking." This could easily be confirmed via western blot to show that you have one band, indicating your tagged protein.



#5 jackster101

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Posted 28 March 2014 - 10:32 AM

So from what I understand--both are driven from diff promoters and the tet response element is only regulating my gene. GFP is constitutively on...



#6 jerryshelly1

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Posted 28 March 2014 - 10:50 AM   Best Answer

Sounds good. Can I get a vector name to satisfy my curiosity?






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