2 replies to this topic

[Richard.21]

Hello everyone!

 

I’ve been facing a problem with my transfection and I would really appreciate your help.

I am trying to transfect a mouse gene into CHO-K1 cells using lipofectamine 2000. My vectors are pcDNA 3.1 Hygro. As transfection efficiency control, I use pcDNA GFP. I transfected the cells in different ratios from 1:1, 1:2, 1:3, 2:2, 2:3.  I usually transfected the cells in the evening and the day after I change the medium in order to avoid cytotoxicity and remove the dead cells. Between two or 3 day after transfection I split the cells and start the selection with Hygromicin B (500µg/ml).

The problem is when I test these cell in PCR they often are positive to my gene but when we try to detect the protein expression by FACS, this is very low (for CHO cells…around 20 or 30%). I don’t know what should I change or do in my transfection protocol.

I sequenced my full vector and it is ok. I sequenced the promoter region and also was ok. I tried Lipofectamine LTX and Roche Fugene HD  that they claim to be less cytotoxic but I had the same results. I heard that the linearized vector the transfection would be worst but the genome integration is better. I tried this procedure and I had the worst results…

I don’t know anymore what should I change or what should do to get high percentage of transfected-expressing cells. This is not normal for CHO-K1 cell.

 

I would really appreciate your opinion

Best Regards

[bob1]

Transfection with these methods is never 100%, the most you will get with a transient transfection is probably around 40%, and the same can be extrapolated to making stable cell lines. 

 

You probably need to start your selection earlier - 24 hours post transfection should work fine.  Make sure that you have titrated the antibiotic to give you the right concentration that will kill all non-tranfected cells.  

 

Transfection does not guarantee that the expression will be high - some (the majority) will be relatively low expression as only a few copies will have integrated, but others will be higher as more copies have integrated.  To choose the high expressers you will need to do single colony isolation and grow them up, then test for expression in each individual line. 

[Olivia Zabel]

Hi,

 

in general all commercial transfection reagents are cationic in charge which leads to aggregation of cells when used in suspesion culture (like CHO cells). This decreases transfection efficiency because not every single cell will be reached.

 

For plasmid transfection of my CHO cells I use a new kind of transfection reagent which is zero in charge and avoids aggregation. Its called Viromer YELLOW and was develeopd from Lipocalyx (www.lipocalyx.de). Viromers are syntehtic polymers and due to their zero charge and an active endosome escape mechanism I get higher transfection efficiency compared to standard reagents.

 

Plasmid transfection of CHO cells by using Viromer YELLOW was published too (see attached file).

Attached Files

•  Biochem Biophys Res Commun.docx   18.65KB   156 downloads

Edited by Olivia Zabel, 19 November 2014 - 01:09 AM.