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retroviral generation of stable cell line


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#1 kokoro

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Posted 21 May 2009 - 02:12 AM

Hello all,

I am trying to produce a cell line stably expressing my protein of interest. Things, however, aren't going too well, and I would appreciate any advice I could get.

so here's what's been happening:

about two weeks ago, I CaPO4 transfected Platinum-A retroviral packaging cells with my plasmid of interest (in pMXs-puro vector).

2 days later I collected the supernatant (which contains the retrovirus).
I applied that to my target cell culture line... after 8 hrs changed medium

2 days later I started selection with puromycin at 1ug/ml (cell culture about 70% confluent)

Selection seemed to be working... cells started to die off gradually about 36hrs after puro addition, and about a week ago, the culture hit an all time low (about 10% confluency). I have changed medium every 2 days since... but after hitting the nadir my culture just seems to have stopped growing.
I change medium at the beginning of the day, check at night, check the next morning, and again at night... and no apparent increase in cell number. What I notice, however, is that there is a lot of cell debree in suspension; when I change medium, for instance, I see the adhered cells, and not too much junk in suspension... but by the end of the day there's a lot of tiny black dots (which I assume is cell debree)

any suggestions... should I just keep waiting?
thank you

#2 madrius1

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Posted 21 May 2009 - 06:19 AM

Seems to me like your virus transduction has not worked. Is it because of the virus production, or the virus efficiency? I couldn't tell. But i've been working with retroviruses for a while and it works perfectly. In my lab though, we use 293T cells for virus production. But the rest of the protocol is pretty much the same : transfection (pAmpho + expression vector), wait for 2-3 days, collect supernatant, add 4 ug/ml polybrene and proceed to the infection. On the day of the infection, the medium from subconfluent cells is removed, then a 50% solution of virus DMEM is added in a minimal volume (e.g. 2ml for a 100mm plate) for 1 hour, and 8 ml of DMEM are added afterwards. Puromycin selection can be done as early as 1 day after transduction, but 2 days are still the best.

Here are some points you may want to check :
- Did you add polybrene to your virus stock?
- Is you virus amphotrophic (is he able to infect your cell line?)
- Were your cells confluent when you infected them?
- Or, if you virus works well and all, is it possible that your protein of interest is toxic when overexpressed?

#3 kokoro

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Posted 21 May 2009 - 10:57 PM

Hi Madriums,

thanks for the prompt reply; I appreciate it

I've gone over what you said, and all I can think of is that maybe my target protein is toxic.
Polybrene was added during infection; the virus is amphotropic; cell confluency wasn't a problem, I think (70% confluent)... just the toxicity issue, it's something that I can't be sure of since it's a first in our lab

the controls I ran - 1) blank (no puro resistance)
2) empty vector (puro resistance there, but not expressing my target protein)
3) target construct

I mean, #1 is gone; #2 is growing a lot better than #3 (likely due to easier insertion into genome - 50~60% confluent); and then, well #3 is what I'm here talking about

I think I will wait 5 days more (that's 2-3 more media changes; and that will be a little over two weeks since puro selection started)
thanks again

#4 kokoro

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Posted 05 June 2009 - 11:11 PM

Hi everyone,

Just wanted to sort of update what has happened, with hopes that I could get a little feedback on what you all think...

So I have been replenishing puro medium every 2 days for the last 2.5 weeks or so... luckily, cells have been growing...
About a week and a half ago I trypsinized the few cells I had (on 6cm plate) and seeded them onto one well (out of a 12 well plate), maybe thinking that concentrating the cells might aid them grow. I think it helped (cell # did increase)... so 4 days after the transfer I again reseeded onto a 6cm dish and have kept them there since.

Now, the cells are growing, but they are doing so very very slowly, so that I can't really do any sort of experiments with them. Moreover, they have changed morphology. We're working with breast cancer cells... in the particular cell line we're using there's the nucleus (imagine it as candy) and say the cytoplasm is the wrapper around it - so that the cells have a nucleus and a stretched thin cytoplasm... you could say they look like a thin eye. Recently though, the cell cytoplasm seems to have grown... morphology is no longer necesarily narrow and long for all cells... some have acquired diamond like shapes...

We're keeping them alive because... well they're surviving puro selection. My supervisor has hopes the phenotypically different cells will eventually die off.

What do you think?

I am not really sure what sort of answer to expect... but ehhh, just have the urge to hear something from someone else, I guess

thanks




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