Can shRNA be used in one transcript with other gene - (Mar/28/2007 )

Hey!

I am wondering if it is possible to use a vector, which inserts my DNA construct into the human cells:

promoter - gene1 - IRES - shRNA(for gene2)

Will this RNA silencing work if it is transfected to human cells?

(shRNA is for Traf1 found in codex database)

What is the best way to measure the efficienty of RNA silencing?

I can think of a sendwitch ELISA, with peroksidase on the 2nd antibody and then measure with luminometry.

Thank you for youu help!

marko

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Do you have a promoter for shRNA, a polymerase III like H1, or U6. This might be better.

I have made
1. promoter-gene1- shRNA-HI promoter
2. H1 promoter - shRNA - promoter - gene1.

Both of them work. You have to check if the promoters (II and III) work in the human cells.

H1 works in human cell line (SHSY5Y).

RT PCR would be quite sensitive to check for silencing.

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First, thanks for your help.

The problem is that I have a LTR promoter from virus HIV:

LTR(HIV) - gene1 - IRES - shRNA

If I make HI promoter in the end, it will not be transcribed only at the time with gene 1. I don't wont that this RNA is transcribed on its own.

How can I solve this? if I use different type of RNA?

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yes, there will be different expresssion levels by different promoters.

I dont understand what type of expression do you want ? if you could explain, we could think up something.

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->I dont understand what type of expression do you want ? if you could explain, we could think up something.

Imagine a transfected cell with a construct based on LTR promoter:

LTR- gene 1 - ires - RNAsilencing

When you inject TAT protein in this cell, then the expression goes up.
So, this means that TAT is a transactivator of our complex.

I would like that gene1 and RNA silencing sequence are expressed together and only when there is TAT present in the cell.

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I don't know if I have been clear enaugh.

I don't wont that shRNA is transcribed in a cell, if I do not inject TAT protein that activates transcription.
And if I put a different promoter this will happen.

Is there a way to fix this?

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How about an inducible system for the shRNA. This should work.

so when you apply TAT, you could induce shRNA expression with chemicals.

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No, unfortunetly this is not an option.

I need to induce transcription of shRNA when I put a minimum virus HIV in the cell. Automaticly, without any other chemicals.

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This is also a possibility:

construct1: LTR-gene1-IRES- gene2

Gene 2 is the activator of a shRNA promoter.

construct2: U6 - shRNA


Then TAT activates construct 1, and gene 2 from construct 1 activates construct 2 and sh RNA is expressed.

Now, I need to solve problems with basal expression of U6 promoter and its activation.

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you will be adding doxycycline thats just another antibiotics. Does that interfere with your experiments?

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