5 replies to this topic

[Help!!]

I have been trying to overexpress my gene of interest into A549 cells with no good result. I have tried playing with the amount of DNA I use to transfect the cells (1, 5, and 10 ugs). I have also tried to use different transfection agents (Lipofectamine, Lipofectamine 2000, Fugene). I have been able to transfect my gene transiently, verifying by Western blot  using an antibody towards my protein and a V5 antibody. I am using pcDNA3.1 with a neomycin selection marker as my vector. I have screened over 50 clones with no real significant overexpression. I have been able to use different vectors other than pcDNA to establish stable cell lines in A549 cells. Can some please help?????

[Helios]

check if your protein is toxic to the cells. it might be that after overexpression,all your transfected cells are getting killed.
if this is not the case,then the integration of your plasmid is such that it disrupts your gene of interest.try linearising your plasmid.it will prevent detection of false positives.you might also want to lower the antibiotic selection in the beginning and then increase it later.
all the best!!

[Help!!]

Helios, on Nov 1 2009, 02:11 PM, said:

check if your protein is toxic to the cells. it might be that after overexpression,all your transfected cells are getting killed.
if this is not the case,then the integration of your plasmid is such that it disrupts your gene of interest.try linearising your plasmid.it will prevent detection of false positives.you might also want to lower the antibiotic selection in the beginning and then increase it later.
all the best!!

Thank you for your suggestions. I will try linerizing my plasmid and transfecting it that way.

[wirly]

Help!!, on Nov 1 2009, 03:27 PM, said:

Helios, on Nov 1 2009, 02:11 PM, said:

check if your protein is toxic to the cells. it might be that after overexpression,all your transfected cells are getting killed.
if this is not the case,then the integration of your plasmid is such that it disrupts your gene of interest.try linearising your plasmid.it will prevent detection of false positives.you might also want to lower the antibiotic selection in the beginning and then increase it later.
all the best!!

Thank you for your suggestions. I will try linerizing my plasmid and transfecting it that way.

All good comments by Helios - can have a 10-100 fold increase in efficiancy.  I'd add that changing your vector may help, though I've found that pcDNA3.1 has a pretty broad spectrum of effectiveness across cell lines.  A different promoter such as EF1 MAY help increase expression.

If you have access to a FCS sorter, you can select for high expressers before you clone them.  This is very powerful technique for getting high expressers..

[Helios]

hey wirly...
can you please elaborate on how to sort cells with the FCS sorter...what if the overexpressing protein doesn't have a fluorescence tag????

[wirly]

Helios, on Nov 10 2009, 09:22 PM, said:

hey wirly...
can you please elaborate on how to sort cells with the FCS sorter...what if the overexpressing protein doesn't have a fluorescence tag????

You know, I was too quick to answer - the FACS sorting (I also mistyped FACS) will only work if your protein is surface expressed - sorry if it's not.  If it is then you can stain it with an antibody+fluorescent tag and sort out the fluorescent cells on a FACS machine like an Aria, but you have to have access to such a machine and a trained operator.