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Problem with expression of marker gene - (Sep/02/2005 )

Hi all... smile.gif
I'm new in this field and I have some problem... unsure.gif

I infect 3t3 cells with lentivirus sups that has GFP marker gene to find out if lentivirus work or not.

I use 6 well plate that already had 3t3 cells ready for infection and I use 3 different volumes of the lentivirus sups 500,250 and 100 microlitter and I bring up to 500 microlitter with DMEM..
For the positive control I use leftover lentivirus sups(already in -70 for 3 weeks) that already tested for expressing GFP in the infected cells before.
For negative control I only put DMEM at 3t3 cells.

after that I put 0.8microlitter polybrene and incubate at 37 degree for 5 hour
and put DMEM again and incubate again for 2 days.

After 2 days I aspirate and wash with pbs and trypsin and put hbss, spin them and resuspend pellet with hbss again..
After that I test them with FACS and the results are all the samples did not have GFP expressed. All of them looked the same as negative control in FACS..

I guessing maybe the infection step that went wrong because my positive control not worked too but I'm not sure about this...
I 'm so confused what went wrong sad.gif ...is anybody know why they not expressing GFP?

Thanks in advanced...

-verencia-

Well something definitly went wrong if your positive control wasn't positive...

Here are several possibilities:

1. Did the lentivirus infection work?

Generally you should see a change in the cells when you infect them with a virus. How did your cells look under the microscope? What do other people in your lab say about how the cells should be looking?

2. Was your FACS analysis correctly set up?

You really need to discuss this with someone who can look at your data. But did you check your cells under a fluorescent microscope before doing the FACS? If they were green then, but did't show anything on the FACS, there's something wrong with the FACS setup. I guess you didn't look under a fluorescent microscope though...

3. How do you know that the supernatants did contain viable lentivirus?

Your positive control could also have suffered from being frozen and defrosted too much



QUOTE (verencia @ Sep 3 2005, 04:41 AM)
Hi all... smile.gif
I'm new in this field and I have some problem... unsure.gif

I infect 3t3 cells with lentivirus sups that has GFP marker gene to find out if lentivirus work or not.

I use 6 well plate that already had 3t3 cells ready for infection and I use 3 different volumes of  the lentivirus sups 500,250 and 100 microlitter and I bring up to 500 microlitter with DMEM..
For the positive control I use leftover lentivirus sups(already in -70 for 3 weeks) that already tested for expressing GFP in the infected cells before.
For negative control I only put DMEM at 3t3 cells.

after that I put 0.8microlitter polybrene and incubate at 37 degree for 5 hour
and put DMEM again and incubate again for 2 days.

After 2 days I aspirate and wash with pbs and trypsin  and put hbss, spin them and resuspend pellet with hbss again..
After that I test them with FACS and the results are all the samples did not have GFP expressed. All of them looked the same as negative control in FACS..

I guessing maybe the infection step that went wrong because my positive control not worked too but I'm not sure about this...
I 'm so confused what went wrong sad.gif ...is anybody know why they not expressing GFP?

Thanks in advanced...

-LeserattePD-

Verenica,

Why are you using the FACS to check expression of your GFP in your 3T3 cells. It's far to complicated, to much work and to many things can go wrong. Just have a look at your (living) cells under a fluorescence microscope. That's all.

-Theo22-