Protocol Online logo
Top : New Forum Archives (2009-): : Protein Expression and Purification

Transient transfection using linear 25 kDa PEI transfection reagent in CHO k1 - (Apr/22/2014 )

For 6 months, I am working on transient transfection of reporter GFP-N1 for evalute transfection efficency. I am using CHO k1 suspension as cell type and polyethylenimine (PEI)as transfection reagent in CHOmacs media. Till today it worked in 96 well plate but not in shake flask. At the ratios from 1:1 till 3.5:1 PEI:DNA. Despite everything is same, I couldnt get repeatable results. other important thing is cell vaibility, it was highly negatively affected and transfection efficiency still very low not more than 10%. therefore what should i do to increase transfection efficiency accompanied by celll viability as well.
I mixed only PEI with DNA dissolved in TE buffer for 30 minutes at room temperature then added to 100 microliter /well. PEI 25 kDa linear. Therefore what should i do to solve PEI cytotoxcity. High PEI amount result in cell death. The maximum amount that I could transfect was 5 micro gram per well (for 96 well plate). Does anyone has idea? What should I do to transfect GFP plasmid using PEI to achevie high transfection efficiency. Please help me. I couldnt cope with alone.

-body2013-

5 ug/well is a massive amount for 96 well plates.  I use less than 0.1 ug/well with my transfections in 96 well plates, and would use a maximum of 2 ug/well for 6 well plates... try using less DNA, too much can lead to massive over-expression which will kill your cells.  Also the lower the amount of PEI you use, the better, so if you can, try using less DNA and hence less PEI.

 

Most transfections work well at a ratio of 3:1 (ul PEI: ug DNA), but you should try the following ratios 1:3, 1:1, 3:1, 6:1.

-bob1-

I am doing serial dilution expriment to detect which concentration could give GFP expression. And that was done by applying fixed concentration 2 ug DNA/well mixed with different amount of linear 25 k Da PEI 2ug , 3ug ,4ug,5ug,6ug,7ug in 200ul cell suspension /well.  100 ul from that transfected cell suspension was transfered to another well that have 100 ul fresh CHOmacs media and so on for 5 times. Therefore 0.1 ug/well concentration was tested.

The transfection work only with the following ratios 1:1, 1.5:1, 2:1,2.5:1 PEI: DNA, at 2 ug DNA/well. but with low transfection efficiency and high toxcity.

PEI dissolved in water was added to DNA dissolved in TE buffer for 25-30 min incubation period.

i use the PEI from Polysciences Inc.,Germany

What is the mximum amount of DNA that you transfect to CHO cells by PEI to acheive high transfection efficiency ?

what about cell vaibility with PEI reagent.

-Body-

-body2013-

Suspension CHOs can be difficult to transfect - have a look around the literature to see what others have done.

-bob1-