help on generation of stable cell lines with pBabe vector - pBabe-puro, phoenix Ampho cells (May/26/2009 )

I was using pBabe-puro to clone the genes of interest (with GFP tag) and got retrovirus with Phoenix Ampho cells. The titer is not high but I got 15-50% HCT116 cells infected based on FACS sorting. After 3-4 round sorting, the percentage of GFP-positive cells remained in the range of 30-70% and the GFP signal is very low. I assume most of infected cells should have the constructs integrated into the genome and become stable. Why didn't I see the stable clones? Also why the GFP expression level is so low (compare to plasmid transfection)?

With plasmid transfection method, I could get over 95% GFP-positive cells after 3-4 rounds of sorting. why retrovirus does not improve? The reason I switched to retrovirus system, is because with plasmid transfection method, some of the stable cell lines later loose the target gene expression (they are still GFP-positive but the tagged target proteins got truncated).

I'm wondering if anyone has more experience with pBabe or other retrovirus vectors and if low expression level is common (due to silencing of the virus promoter?) It could be due to the specific mutations I introduced into the target gene, which either cause protein instability or toxic to the cells.

Retrovirus mediated transgenesis usually has lower expression level than transfection, because each cell can receive few copies of the transgene.
The non-expressing cells in the sorted cells can easily outgrowth the population. You may have better pick clones, or increase the moi. I used moi=10 and got ~100% expression.

WHR on May 27 2009, 12:08 AM said:

Thanks for the reply. It helps to know low expression level is intrinsic to retrovirus mediated infection. What I don't understand is that after each sorting, GFP-positive cells are over 98%. then I kept growing them for one more week for another round sorting. If they are truly integrated into the genome, the non-expressing cells should not overgrow the population to such a high percentage within one week. Unless the GFP-positive cells also keep losing signals. I will try to pick single clones for the analysis.

Why don't you just select clones with puromycin?

It's really fast and effective!

if you sorted your cells and got 90% GFP positive and a week later they are all negative it sounds like it might have to do with your gene of interest rather than with the method.
(or the FACS run was not under the same conditions as the first run)

are you sure that you actually got GFP+ cells and not autofluorescent dying cells in your first sort?

Is your GFP a fusion or an iRES? It could be that the cells don't like your gene and therefore silence or otherwise inactivate it.

It can also be that your GFP positive genes just stopped dividing due to the gene of interest - if your cells divide once per 24h it does not take too long to outgrow a population:

starting from
10% negative 90% positive

assuming that the positive stop growing, will give you

93% negative 7% positive in 7 days.

warsel on May 27 2009, 12:45 PM said:

Here are the percentage of GFP-positive cells after each round of sorting:
15%, 50%, 75%, 30%,

It's a GFP-fusion protein. Those GFP-positive cells are not dying. Since I don't have single clones I can't really compare the growth rate of GFP-positive and negative cells. Someone told me that GFP sorting is a more effective and fast way to enrich cells than puromycin selection.

I'm wondering in your hand with you target genes, how fast you can get >95% GFP-positive cells and how stable they could be. Do you see significant improvement with retrovirus than with plasmid transfection when generating stable clones?

tsoimp on May 27 2009, 09:17 AM said:

Hmm.. you only got 15% GFP+ cells post sort - or am I getting you wrong? This seems very low..

I much prefer puromycin to sorting as well - you add puromycin the day after infection/transfection and keep it on for 1-3 days that's it.
So, answering your question, I usually have >95% GFP+ cells after 2-3 days post transduction (regardless of if I'm using tranfection, lentivirus or retrovirus).

Lenti- or Retroviral is much more effective than transfections for stable cell generation but it also takes a little more time to do (if you don't have the virus particles already).

warsel on May 28 2009, 09:06 AM said:

I got 15% positive when I first did sorting (2 days after infection). Right after sorting, it's >95% positive. I let them grow for one week. It became 50% positive when I did the second time sorting.....It reaches to 75% in the third time sorting (the third week) and goes down to 30% in the fourth time (or the fourth week). It seems now that the problem is largely due to the particular mutations introduced in the gene. I will definitely try selection this time.