Transfection of human fibroblasts - (Apr/09/2008 )
We had a lot of troubles with transfection of our fibroblast celline.
We tried Lipofectamine 2000 and fugen.
But after transfection al the cells died.
And we have a very low transfection efficiency.
The construct contains the human promoter and Egfp followd by our gene of interesting.
Can somebody help us? Becouse we don't have any idease anymore to transfect.
Thanks!!
Carola
Hello ,
In regrading the cells die , if the cells die 24 hrs post-tranfection ,that mean you have added high amounts or concentration of lipofectamine 2000 because lipofecatmine is toxic to the cells unless you optimize your concentration.How much did you use ,which kind of plate did you do your transfection and howmuch DNA have you used.Another thing you should also undertaking the amount of DNA ,sometimes if you use much DNA that might killed the cells.In concern you have got very littel expression ,that could be your plasmid ,try to look into your plasmid map and see what kind of promoter does your plasmid have.Moreover ,look to the site of your gene insertion and your opening fram ,otherwise you will not get enough protein ,look also into your restriction site may you inserted wrong,have you squenced your gene before insertion.On the other hand, be careful to look into your gene from the start codon ,I mean from the begining of your gene sequences has your gene contain AUG as started codon .
Ela
With the human skin fibroblasts that we use only lentiviral infection gives efficient infection without cell death. For transfection, even though with less efficiency, Fugene-6 performs better than Lipofectamine-2000.
I would optimize transfection first with any reporter plasmid. My exeprince manily with fugene and I found when you use high amount of either DNA or Fugene you kill the cells or have low transfection
In regrading the cells die , if the cells die 24 hrs post-tranfection ,that mean you have added high amounts or concentration of lipofectamine 2000 because lipofecatmine is toxic to the cells unless you optimize your concentration.How much did you use ,which kind of plate did you do your transfection and howmuch DNA have you used.Another thing you should also undertaking the amount of DNA ,sometimes if you use much DNA that might killed the cells.In concern you have got very littel expression ,that could be your plasmid ,try to look into your plasmid map and see what kind of promoter does your plasmid have.Moreover ,look to the site of your gene insertion and your opening fram ,otherwise you will not get enough protein ,look also into your restriction site may you inserted wrong,have you squenced your gene before insertion.On the other hand, be careful to look into your gene from the start codon ,I mean from the begining of your gene sequences has your gene contain AUG as started codon .
Ela
We started with 10 ul lipofecamine 2000, and we reduced this into 5 ul lipofecamine 2000 per well of a 6-wells plate.
And we added 0,4 ug plasmid.
Our construct contains a human promoter, and we sequenced the gene complete.
In a cos-cells transfection we confirmed that the construct works well.
We tried an other construct, which is used by other people in our group, and with that construct our cells will die also.
So maybe the problem is the lipofecamine. But how less amount lipofectamine can we use?
Hello again,
Ok now everything clear , your DNA is too low ,first increase your DNA , , your DNA should be between 2.5 to 10 ug.For lipofectamine it is ok but you can use 2.5 to 5ul.You can do transfection in OptiMEM or any medium reduced serum for 24 hr ,if your cells die ,try to reduce time of transfection or transfect in OptiMEM contain 1-2% serum for 8-10 hrs ,thereafter replace the medium into complete medium for 24 hrs ,then assay your gene expression .You should assay your gene in medium serum free.
Good luck.
Ela