8 replies to this topic

[gyma]

Hi there.
I am now going to inject leukemia cells to nude mouse to see tumor formation. I have one target gene knockdown in this cell line by lentiviral shRNA expression plasmid. I have confirmed the knockdown effect and sorted 95% GFP-positive cells. I was told that a stable transfectant is needed for injection to nude mouse but unfortunately I dont have a drug-resistant gene in that plasmid. So my questions are:

1. How should I get stable transfectant without drug-resistant gene in the plasmid?

2. The target gene might be essential for leukemia cell growth. In that case, can I get stable transfectants if it is indispensable?

3. Normally lentiviral transduction of foreign genes will give a longer expression than other transient transfection methods, lipofectamine e.g. But how long?

4. And I observed that the fluorescence coming from empty vector always sustains longer compared with the one with insert. It also happens to other lab members and seems to be a common case. Do you know why this is happening?

Thank you.

[tea-test]

you could do single cell dilutions and then select the GFP poasitive clones. That would be my first thought. i assume these are not adherent cells (leukemia)?

[bob1]

It is unlikely that you will get a stable cell line without selection - cells are very good at getting rid of unnecessary DNA.  However, if you continuously culture and select for the GFP expressing line (by FACS?), eventually you may get a stably transfected cell line.

If you are knocking down an essential gene with your transfection, then it is very very unlikely that you will get a stable line out of it unless the shRNA is inducible, such as using the TET-on/off system.

[gyma]

tea-test, on 02 May 2011 - 03:50 AM, said:

you could do single cell dilutions and then select the GFP poasitive clones. That would be my first thought. i assume these are not adherent cells (leukemia)?

thank you. these are suspension cells and dont grow well or even dont grow at all in a low density. So if I get it too diluted to get single cells, even if it is GFP positive, maybe it will not grow. is it not the case in adherent cells?

[gyma]

bob1, on 02 May 2011 - 04:04 PM, said:

It is unlikely that you will get a stable cell line without selection - cells are very good at getting rid of unnecessary DNA.  However, if you continuously culture and select for the GFP expressing line (by FACS?), eventually you may get a stably transfected cell line.

If you are knocking down an essential gene with your transfection, then it is very very unlikely that you will get a stable line out of it unless the shRNA is inducible, such as using the TET-on/off system.

Thank you. I saw people cotransfect another plasmid with selection marker and select the GFP positive cells. does that work?

[bob1]

Sort of, people do use it, and it can work, but there is still no selection for the other plasmid, so it might get degraded or methylated, while the selection plasmid is maintained.

[gyma]

bob1, on 04 May 2011 - 02:09 PM, said:

Sort of, people do use it, and it can work, but there is still no selection for the other plasmid, so it might get degraded or methylated, while the selection plasmid is maintained.

you mean there is chance that cell with both plasmids would be selected but eventually the one with no antibiotics-resistant gene will be silenced, right?
I have sorted transient transfected cells on hand but the expression of GFP decrease dramatically 1 week after thawing. Before freezing it was >90% strong positive, now it is still 90% but with only a slight shift in FACS histogram. Is it because of freezing/thawing or just a common case of transcient transfection?

[bob1]

Transient transfections result in the DNA being degraded and/or methylated, occasionally they will result in a stable cell, but this is very rare and usually isn't detected.  Freezing and thawing activate a whole range of genes known as heat and cold shock genes.  These are known to degrade damaged DNA.  It is not worth freezing transient transfections, as they normally only last about a week before there is no longer any expression of the transfected plasmid.

[gyma]

bob1, on 07 May 2011 - 03:36 PM, said:

Transient transfections result in the DNA being degraded and/or methylated, occasionally they will result in a stable cell, but this is very rare and usually isn't detected.  Freezing and thawing activate a whole range of genes known as heat and cold shock genes.  These are known to degrade damaged DNA.  It is not worth freezing transient transfections, as they normally only last about a week before there is no longer any expression of the transfected plasmid.

Thank you very much. But in my case, I used lentiviral transduction and the fluorescence lasts longer than 1 week. Anyway, since it doesnt work with thawed cells, I have to re-transduce cells. Many thanks.