Recently, I designed an expression cassette.the cassette contains tissue specific enhancer and promoter.Also, 5 prime UTR and poly A signal.Then I sub-cloned my desired insert into the cassette.It is all in pGH (cloning vector).Is it necessary to sub-clone the final construct into the expression plasmid?the point is I have to test my expression cassette.
Thanks.
Hi Shahrooz,
In the most basic sense, you have made an expression vector. So here is no absolute need to sub-clone the expression cassette into another vector.
There are some caveats though: what you have will only work for transient expression, if you want to establish stable lines then you will need further components to supply some selection. If you require any additional components for the promoter to function, they will be lacking as well. Further check that you have a proper kozak sequence at the start of you gene.
Also to test your vector I'd use some genes that you know express well and have a good reporter assay, EGFP would be an obvious choice, before using anything that is specific to your investigations.
-Richard
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