Hi Mohammad.

Check the similar discussion for the construction of vector and necessary components with a simple vector allow transcript expression and GFP translation together.

https://www.researchgate.net/post/How_and_where_can_I_get_the_entire_genome_sequence_of_a_miRNA

Reagrds

Hi @Sang Ho Lee

please read my answer here:

https://www.researchgate.net/post/How_and_where_can_I_get_the_entire_genome_sequence_of_a_miRNA

Hi Mohammad.

1) Why you are putting GFP? That is a role of reporter assuring that your premiRNA has been activated to make its sequences. Therefore GFP is an indicator for your robust expression of premiRNA. The IRES has been adopted for many thousand of gene manipulation studies without fail.

2) Most of miRNA working place is cytoplasm except for reentry tp the nucleus and other organelles in non-conventional miRNAs. However, most of miRNAs are involved in posttranscritional regulation against mature mRNA in the cytoplasm. So, It's generally accepted that they are working in the cytoplasm.

Regards.

Hi @Sang Ho Lee

thanks for reply

Since IRES is the ribosomal landing site. It is used to express two proteins (bicistronic mRNA) with a promoter and a polyA-signal but about miRNA, because we do not have protein expression, so what can IRES do?

Hi, Mohammad.

It would do no harm to your purpose of premiRNA transcript procesed to exporting to cytoplasm and will be dissociated within the the ctyoplasm. There is always possibility that the upstream and downstream of premiRNA sequences may not be exhaustively verified and will be updated all the time. The IRES may be considered as a spacer and is recognized as two genes (target and GFP) are activated at transcription level by RT-PCR and at translation level by Western blot in many studies. You may have in the plan of your BAC construct. In your construct you may have a small fragment to give a space after the GFP termination. But GFP expression would not guarantee your premiRNA until you do RT-PCR.

Regards.

Thank you Dr. Lee for reply

so I think we have 2 scenario:

--------------------------------------------------1----------------------------------------------------------

1: (5'-pB--promoter--chimeric intron-premiRNA-IRES-GFP--polyA--pB-3')

"As you mentioned GFP is a reporter to ensure the expression of the premiRNA"

1A: After splicing we have a mature mRNA with below sequence:

(5'cap-premiRNA-IRES-GFP-polyA-AAAAAA3')

1B: This mRNA will be transferred from the nucleus to the cytoplasm.

1C: After that DICER will cut premiRNA so we'll have such RNAs:

(5'IRES-GFP-polyA-AAAAA3') + (mature miRNA)

Which seems to be both(premiRNA&GFP) functional.Of course, it's probably unstable because of lack of 5' cap!!

--------------------------------------------------2----------------------------------------------------------

2:(5'-pB--promoter--chimeric intron--GFP-IRES-premiRNA-polyA--pB-3')

Everything will look like the first scenario, except for the cut off stage by DICER that we have such RNAs:

(5'cap-GFP-IRES3') + (mature miRNA)

so, first mRNA will be without a polyA-tail and it does not look good!!

What do you think of these scenarios?

Is there any other way??

Hi Mohammad.

I would go with the first vector construction. Once you see the GFP expression, and then assay for qPCR for miRNA expression. At least you could eliminate the cells transfected, but not showing GFP expression for cell cloning.

Best wishes.