I am doing transfection of BHK 21 cells using Lipofectamine LTX plus. My vector is pVAX1, but I am not getting the expressed protein I included kozak sequence before my gene. The protocol which i am following is:
In a 6 well plate after 90% confluence of cells, the media is removed and lipofectamine DNA complex is added (lipofectamine 8 ul in 150 ul DMEM and plasmid DNA 8000 ng in 175 ul of DMEM and plus reagent 3 ul), mixed and incubated for 5 min at RT. Then transfection..
After 4 hours 2 ml of DMEM is added, followed by 48 hours of incubation.
Can anyone suggest what the solution is?
As Edouard says, you need to have a reporter gene like GFP to confirm that you actually are getting transfection. BHK-21 is a kidney cell line, so it shouldn't be that difficult to transfect, especially with BHK-21 specific transfection reagents (https://altogen.com/product/bhk-21-transfection-reagent-kidney-cells-ccl-10/) but you can still end up with lapses in transfection efficiency if your cargo is too large. For that we use complex condensers, but those can sometimes lower the overall transfection efficiency.
Hello,
Do you have a reporter plasmid like pMax-GFP to ensure that trnafection is well operated ? Then if so, you have to check your DNA and RNA sequence. Koazk consensus is not enough, you have to check 5' and 3'UTR sequences.
Sincerely
Dear Elamurugan,
I totally agree with Edourd, it should more efficient to settle your conditions with a control plasmid.
Nevertheless, I would also recommend trying an alternative to LTX that shown some efficiency with BHK cells. You can refer to Pittayakhajonwut D. et al, Virol J. 2010 Jun 10;7:123; the authors used DreamFect reagent to deliver DNA into BHK-21 cells.
for your convinience here is the pubmed link: http://www.ncbi.nlm.nih.gov/pubmed/20537170
The other option is to enhance the LTX efficiency: to use Magnetofection in association with this lipid-based reagent. Magnetic nanoparticles can be added to LTX-DNA complexes and form magnetic complexes that are attracted onto the cell surface under the influence of a magnetic field. CombiMag potentially enahnce transfection efficiency of any commercial transfection reagent.
Would you need any information, please do not hesitate to contact me directly at: [email protected]
good luck with your experiment,
best regards,
Cedric
As Edouard says, you need to have a reporter gene like GFP to confirm that you actually are getting transfection. BHK-21 is a kidney cell line, so it shouldn't be that difficult to transfect, especially with BHK-21 specific transfection reagents (https://altogen.com/product/bhk-21-transfection-reagent-kidney-cells-ccl-10/) but you can still end up with lapses in transfection efficiency if your cargo is too large. For that we use complex condensers, but those can sometimes lower the overall transfection efficiency.
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