I synthesised pAdenovirus expressing Protein-FLAG tagged and reconstituted in knockdown cells. When I perform the experiment, WT cells can perform the gene expression but reconstituted cells can restore only 50% of it (Real Time data).
I have checked at various concentration of the pAd virus but failed to restore the same level. Is this normal or any problem. Please suggest. Thanks in advance!
Hi Pratibha,
expression is always a complicate matter. to answer exhaustively I would need more information for example about promoter used to drive expression from the transgene, if you cloned only the ORF etc.
Remember that the rate of transcription and translation of an endogenous gene depends on signals present on the DNA sequence (i.e. promoter, possible enhancers, etc...) and/or on the mRNA (Kozak etc). when you transfect/transduce a cloned sequence you usually have a viral promoter and downstream you cloned the mature mRNA. Therefore you expect the protein to be produced, but not necessarily at the same level.
Hi Pratibha,
I agree with Paola that there are many details to consider and it could be helpful if you could share more details. Overall 50 % of the RNA is not so bad. It is also not clear to me, whether your knockdown approach could have an effect on your transgene. If you plan a rescue of the knockdown effect the protein level will is important and your transfected construct could give rise to even more protein compared to the endogenous level.
Probably your FLAG tag is altering the stability of your protein or your transformation efficiency is low. You may try generating a stable cell line expressing your chromosomally integrated FLAG-tagged protein.
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