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HMEC-1 stable transfection: blasticidin resistance?

HMEC-1 stable transfection: blasticidin resistance? - Transfection Forum

HMEC-1 stable transfection: blasticidin resistance? - Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


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Old 03-15-2011, 12:36 PM
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Default HMEC-1 stable transfection: blasticidin resistance?



Hi everybody!
I tried to stabelly transfect HMEC-1 cells (a human microvascular endothelial cell line) with a retroviral vector containing the blasticidin resistance gene, but the negative control survived selection. Do HMEC-1 already have blasticin resistance? I couldn't find anything about it in the literature, so I thought maybe someone on this forum might be able to help.....please?
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Old 07-25-2011, 03:00 PM
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Default Re: HMEC-1 stable transfection: blasticidin resistance?

Hi,

I saw your post asking about the blasticidin resistance of HMEC-1 cells. As I'm planning on using these in a couple of weeks (with a vector that has a blasticidin resistance gene), I was wondering whether you had solved your problem? If so, could you tell me which concentration of antibiotic you use? I will still titrate this for my cells, but at least then I know an approximate value to start from.

Thanks,
dpo
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Old 07-26-2011, 09:08 AM
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Default Re: HMEC-1 stable transfection: blasticidin resistance?

Hi!

No, I have not found a solution to the problem. I have, however, meanwhile contacted the group which first immortalized them and atthey told me that they didn't use blasticin-selection, so in principle the cell line should not be resistant.

The only advice I can give you is to start pretty high!

If you find a suitable concentration in your titration experiments, would you mind telling me (I would repeat the titration anyway)?

Regards,

kraefzke

Last edited by kraefzke; 07-26-2011 at 09:09 AM.
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Old 07-27-2011, 09:31 AM
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Default Re: HMEC-1 stable transfection: blasticidin resistance?

Damn, I was hoping that you had found a solution. Anyway, I will start my titration at the end of this week, so hopefully I will have an answer by the end of next week. I'll keep you updated!
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Old 08-04-2011, 06:47 AM
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Default Re: HMEC-1 stable transfection: blasticidin resistance?

So, I added the antibiotic on Sunday and checked the cells today. In the well with 4 g/ml there were just a few cells left, but in the well with 6 g/ml, everything was dead. So I will use this concentration to select my transduced cells.

The blasticidin I used is from Invitrogen, dissolved in distilled water at 5 mg/ml. The HMEC cells were directly obtained from the people who published them (several years ago). I would recommend testing your cells again and simultaneously try out your blasticidin on a cell line for which you know it is sensitive to blasticidin.

In case you would want to change antibiotic, I also tested for puromycin resistance and, similar to a lot of publications, the cells are all dead after four days with 250 ng/ml.

Good luck!
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