Lentivirus copy number - (Jul/08/2014 )

Hi,

 

I recently designed a lentivirus using a cytokine promoter fueling an IRES-eGFP to monitor intracellular cytokine activity, with the end goal to flow sort out GFPhi/low populations representing high/low cytokine activity for downstream in vivo applications.

 

Particularly, I am concerned about lentiviral copy number as 1) extra copies of lentivirus could give false positive, and vice versa, and 2) extra copies of lentiviral cytokine promoter could deplete transcription factors, artificially lowering cytokine levels.

 

I am currently looking into this issue by quantifying a sorted population via ELISA and hoping that what I get matches well with the flow cytometry data.

 

 

To address this issue, I was thinking about introducing a constitutive promoter-reporter pair into my lentivirus plasmid specifically to monitor copy number. However, this results in a clunky plasmid both in terms of lentivirus production as well as downstream flow cytometry applications (2 channels wasted).

 

So does anyone here have some clever ideas on how to monitor copy number while preserving the possibility of downstream applications? I have very little knowledge of plasmid constructs, but I guess my ideal would be a selectable plasmid (i.e. high copy numbers increase sensitivity to some drug, low copy number has no/low effects on viability). Any suggestions?

 

Even if my ELISA results validate my construct, I think this would still be valuable information for me.

quantitative PCR will be the easiest way. You should be able to use this to correlate to fluorescence intensity  if the intensity is related to the copy number.  Ideally you would do this off a part of the plasmid that is not related to the IRES products.

bob1 on Tue Jul 8 22:59:42 2014 said:

quantitative PCR will be the easiest way. You should be able to use this to correlate to fluorescence intensity  if the intensity is related to the copy number.  Ideally you would do this off a part of the plasmid that is not related to the IRES products.

 

Yes, I neglected to mention that I am going to do functional analysis with ELISA as well as qPCR for quality control purposes. Ideally, both turn out good and my construct becomes valid, but I rarely ever have such good luck.

 

My question was if there was a way to select for copy number a priori. qPCR is the gold standard but won't help me fix anything if something turns out wrong.

 

 

Also, why would you not do this off of the IRES products? I conveniently have the primers already for the promoter-IRES-eGFP construct. Is there some DNA interaction that I'm missing?

You could work off the IRES based sequences for correlation with fl intensity, but these are usually under control of very strong promoters so the fluorescence intensity is usually pretty high and won't show much variation.  I would work off a lower expression, different colour, reporter insert if I was doing this. 

 

I don't know of any method to select a priori other than establishing a reliable baseline/correlation curve before you start your true experiments, and then using this to work out the selection level.  That's why I suggested correlation of qPCR with fluorescence.

If you do your transduction (I'm assuming you're doing this in cell culture), you can wait until you see expression of your marker (GFP in this case), split the cells and then plate them at low concentrations.  From there, you can do clonal selection in which case with each cell in a colony will have the same number of copies as the original clone (assuming no subsequent chromosomal rearrangements).  After you have clones, you can do qPCR on a prep from each clone you want to test while keeping the rest safely growing/cryopreserved.  Ideally, if you do different treatments on cells decended from a single clone, the copy numbers shouldn't be a problem...ideally.  That's the only thing that comes to mind here.

Bio-Lad on Thu Jul 10 19:39:04 2014 said:

If you do your transduction (I'm assuming you're doing this in cell culture), you can wait until you see expression of your marker (GFP in this case), split the cells and then plate them at low concentrations. From there, you can do clonal selection in which case with each cell in a colony will have the same number of copies as the original clone (assuming no subsequent chromosomal rearrangements). After you have clones, you can do qPCR on a prep from each clone you want to test while keeping the rest safely growing/cryopreserved. Ideally, if you do different treatments on cells decended from a single clone, the copy numbers shouldn't be a problem...ideally. That's the only thing that comes to mind here.

Unfortunately, this isn't viable as we are trying to separate a heterozygous population via cytokine expression, and this would definitely interfere with that.

I think I will have to do the double expression way.


Thanks, all.

So this may be another possibility, have you considered not doing the lentivirus at all and using a Cas-based homologous recombination approach?  The off-site insertions are on the low side if you do it well and by a targeting specific locus for insertion you could expect an ideal maximum of two copies (or one copy if your cells come from a male and you target the X-chromosome for recombination) per fluorescent cell.

Bio-Lad on Thu Jul 10 20:40:14 2014 said:

So this may be another possibility, have you considered not doing the lentivirus at all and using a Cas-based homologous recombination approach? The off-site insertions are on the low side if you do it well and by a targeting specific locus for insertion you could expect an ideal maximum of two copies (or one copy if your cells come from a male and you target the X-chromosome for recombination) per fluorescent cell.

Unofrtunately, after the boss heard my reasoning, he prefers the secondary reporter way in order to achieve a very tight control on copy number.

If this doesn't work somehow, we might come back to alternative methods. But for now, I'm stuck.