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The right lentivirus plasmid for overexpression in human cells


Best Answer 5280 , 28 February 2015 - 11:40 PM

Inducible expression is better. You can fine tune the expression. Also lentivirus is more efficient than retrovirus. pCW57.1 works well for Dox inducible expression of your GOI. You can package lentovirus with pspax2 and pmd2.g. If you want inducible knockdown with shrna then try pLKO tet on. Go to the full post


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#1 TheFOrsh

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Posted 28 February 2015 - 06:21 AM

Hi, I was wondering if anyone could recommend me or perhaps suggest to be a good plasmid for overexpression of a gene in human cells?

 

I want to create a stable line of neuroblastoma cells (SH-SY5Y and/or IMR-32) and endothelial cells (hBMEC, TY10, or CaCO-2) which overexpress my human gene of interest and i'd like to use lentiviral transduction to get the best efficiency.  I also would like the gene untagged as I'd like to observe normal function when upregulated.  

 

From a literature review i've decided that the most important thing is going to be choosing the promoter.  I would prefer to select by puro/neo/blast etc rather than using a cell sorter and fluorescent marker protein, but either would be OK, I would also prefer it if the cells didn't express any fluorescent protein at all because i'd rather they don't transcribe anything unnecessary - but i'm not overly fussed.  The most important thing is the right promoter.

 

So i've gathered that CMV is a general 'one-size-fits-all' and works for most things, but that some promoters may work better for specific cell lines.  The U6 promoter is reported to work really well with neuroblastoma cells, while CMV is more modest in action.  CMV seems a pretty good fit for endothelial cell lines, though I did find a paper that found U6 was really good in the endothelial cells of mice.  One thing i'm particularly interested in is possibly using tet-on overexpression, has anyone found this to be effective in the cell lines i've described?  If tet-on is an effective promoter then that would definitely be the best choice for me.  

 

Can anyone recommend me a plasmid they have used which is effective? So far i've narrowed it down to pLJM1-EGFP (CMV), and pCW57.1 (tet-on).  I've not been able to find a U6 promoter that isn't for shRNA that isn't from a commercial company and extremely expensive.

 

Most importantly .. am I going the right way if I want to run experiments to see the effect of upregulation of my gene on cell function?

 

 

Thanks for reading.

 

:EDIT: I should add - the backbone should be 2nd or preferably 3rd generation because of safety concerns.  Otherwise I would use pBABE as it seems widely used, though appears to not be 2nd/3rd gen and produces viral particles without needing to be transfected with packaging plasmids. 


Edited by TheFOrsh, 28 February 2015 - 07:45 AM.


#2 5280

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Posted 28 February 2015 - 11:40 PM   Best Answer

Inducible expression is better. You can fine tune the expression. Also lentivirus is more efficient than retrovirus. pCW57.1 works well for Dox inducible expression of your GOI. You can package lentovirus with pspax2 and pmd2.g. If you want inducible knockdown with shrna then try pLKO tet on.

#3 TheFOrsh

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Posted 01 March 2015 - 03:50 AM

Thanks 5208, i'll give pCW57.1 a go.  It has a gateway cloning site too which is handy, though they don't seem to be mapped properly on the plasmid map.  Another group working with our GOI is sending us their own pLKO-tet-on shRNA plasmids which is handy!



#4 5280

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Posted 01 March 2015 - 02:42 PM

FYI, the puro resistance gene in tet-pLKO is downstream of an IRES => hPGK (promoter)-TetR-IRES-puro. This means expression of puro resistance is not as high as it would be if expressed by its own promoter or downstream of a 2A self cleaving peptide such as with pCW57. You may need to select your cells with a lower puro concentration with tet-pLKO (shrna)as compared to pCW57 (goi). Make sure your using clontechs tet free fbs when you start working with these Dox inducible systems.
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#5 miST32

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Posted 19 February 2016 - 12:11 PM

Bumping this thread to see how the pCW57 system has worked out for people so far.  How tight has the repression been in the absence of dox?

There is some data in the literature, but I'm curious how it's worked for others.



#6 5280

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Posted 18 March 2016 - 12:05 PM

I do not see induction in no dox condition for transduced cells as measured by fluorescent with RFP or GPF, RT-PCR and Western blot. Using a more sensitive method, you may be able to detect some low level non-specific induction with a luciferase assay (pCW57.1-luc). I have seen induction in the absence of dox for transient transfection in 293T cells. These cells transfect very well and probably get a lot of plasmid which is why this low level non-specific induction is now detectable. 



#7 miST32

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Posted 18 March 2016 - 02:17 PM

Thanks for the update - We just got our system up and running for initial characterization last week.  So far so good.

I have a bit of leaky expression detected by western blot in my mixed/polyclonal cultures, but at least 10-fold increase in expression after dox treatment.  No detectable fluorescence without dox.  I'm quite pleased!

I suspect that the leaky expression has to do with a few cells that were infected with more virus than others, and that we'll be able to find a suitable single cell clone with near zero basal expression.



#8 5280

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Posted 18 March 2016 - 04:07 PM

You may see this low level of leaky expression if you transduce cells with complete supernatant, especially if you have high viral titers. Like you said, some cells can be transduced with multiple viral copies. Not sure if you're doing this, but if your getting efficient virus product then you should transduce cells with complete sup, serial dilute 1:3 or 1:5 over about 5-6 wells and finally select with puro at 48 hours post transduction. Lastly select the well where you see about ~30% of the cell surviving. You can also take this well and split it to a larger dish so you can pick single clones. Most should have good induction without the leaky expression. 


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