Since you are using the cnstruct for expression in mammalian cells, you need to supply T7 polymerase to the cells for initiation of transcription, inside the cells. This can be done using T7 pol constructs which express in mammalian cells or through infection of the host cells by adenovirus carrying the T7 pol genetic constructs. For reference please go thrugh the following paper:

Article Construction of an eGFP Expression Plasmid under Control of ...

best wishes,

SD

agree with @ Sibnarayan Datta

regards

Follow this link

Article Improvement of reporter activity by IRES-mediated polycistro...

regards

Dear All, thanks for your reply

i would like to clarify again that i am using in vitro transcribed RNA by taking vector with insert as template . The final RNA theoretically have IRES upstream to coding sequence followed by 30 nucleotide poly A tail, this mRNA is transfecetd to HEK cells. I expecetd GFP signal after transfecteion, which is missing there.

few points on which m working are:

1 adding cap to increase stability from exonuclease

2. increasing length of poly A tail

3. adding modified nucleotide such as pseudourdine and 5 methyl cytosine to enhance stability

4. using mRNA specific transfection reagent ( messengerMAX)

Kindly let me know what else i can try or mistake m making

As I have earlier mentioned, I again repeat that HEK cells do not generate T7 polymerase. Since you GFP is under T7 promoter, it will not be transcribed until T7 polymerase is provided. You can provide T7 pol through co-transfection of another contruct that expressT7 pol under a mammalian promoter. IRES is a translation initiation element, that comes into picture only after transcription. If you dont want to co-transfect another constuct, then replace T7 promoter with another promoter, that can initiate transcription of the GFP construct in HEK cells.

Hope you got my point. You may refer to similar papers also.

best of luck,

SD

I am attaching our previous work with HCV IRES -GFP construct in Huh7 cells, where we have infected the cells with an adenovirus ( AdexCAT7 ) containing T7 RNA polymerase.

http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0072791&type=printable

SD

Dear Sibnarayan

thank you for taking interest in my query and i appreciate you quick response . but my problem is different . i am transfecting RNA not DNA. It is already transcibed outside the cell by T7 polymerase during vitro transcription. so HEK need not to have T7 polymerase because RNA is being transfected not DNA vector. plz correct if m wrong

OK Sanjesh, I got your point now. You have already passed all the necessary check-points, such as sequencing, RNA integrity, quantity etc. So now you have to confirm that transfection is successful. Please check all the necessary protocols for transfection. especially check the media composition and volume recommended for transfection. Transfection is usually done in media containing lesser percentage of FBS/FCS and in a very minimal volume, which depends upon your culture plate type (6 well or 12 well). Follow the recommendations of the transfection reagent vendor. If everything is right, and still you are unable to get signal then you may consider trying another transfection reagent such as Xfect, RiboJuice, TransIT, etc.

Next, you also have to carefully check your fluorescence microscope settings and have to be sure that you are using correct filter settings for the particular GFP variant that you are looking for. Many a times expression of GFP is very faint and may not be detectable. In that case, you can confirm expression by western blotting with anti-GFP.

Another important point- please check the source of your GFP coding region and be sure that your GFP reading frame is codon optimized for efficient expression in human cells.

best wishes,

SD

Article Selection of an optimal RNA transfection reagent and compari...

Dear Sibnarayan ,

Thank you for your suggestion. i have HIS tag in the frame of GFP at 3'end , i will try western blotting using anti HIS antibody.

Is it possible to get any T7 polymerase expressing mammalian vector from your laboratory, which i can co-transfect with my current DNA plasmid to confirm the expression from my construct, As my DNA transfection is well optimized.

I have friend in Tejpur university who can collect , if it is available with you

my e mail ID [email protected]

Dear Sanjesh, I am sorry to inform you that I am presently not having any T7 expressing construct. I used the construct during my postdoc almost 9 years back.

SD