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If your goal is to cause a specific protein to be expressed transiently via an episomally maintained gene, could you just exclude all introns and the exons not associated with that transcript variant when choosing what DNA to package into your plasmid for that gene? So far as I know, the role of introns is usually in allowing alternative splicing and in modifying gene expression. But if you're only interested in one transcript variant, you wouldn't need alternative splicing to occur on the episome, and gene expression is handled in plasmid design by the selected promoters and enhancers.

For those reasons, could the introns (and exons not used for that transcript variant) be excluded? And would that affect the protein produced?

Gumpf
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    Why don't you use a mRNA transcript for expression cloning? – Chris Oct 15 '23 at 17:53
  • @Chris Sorry, could you elaborate a bit more? When you say mRNA transcript, would you just put the DNA complement of that mRNA into the expression vector? – Gumpf Oct 15 '23 at 18:56
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    Exactly. I can write you a more complete answer about this tomorrow. Basically you "use" the mRNA which has the exact sequence of the expressed gene. Avoids you doing a ton of edits and possible mistakes. – Chris Oct 15 '23 at 19:31
  • @Chris I would appreciate that! Your comments give me some threads to pull at with reading on my own too in the meantime, thank you! – Gumpf Oct 15 '23 at 20:25
  • @Chris If you pulled the transcript variant's mRNA from NCBI or similar, would you just form the complement from the sequence listed there? Or is it already "complemented" where that sequence is what you'd want going in the same direction as your other plasmid components? And could you just use the CDS with a custom promoter like EF1a? Or would you typically use the wholesale sequence from NCBI? – Gumpf Oct 15 '23 at 22:24

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Use the coding sequence from the mRNA form available on sites such as NCBI

The sequences on NCBI etc are already in the DNA form; note the absence of any Uracils, indicating that this isn't actually mRNA as such, but rather a DNA copy of the RNA.

These sequences often contain 5' and 3' UTRs, so to be usable for you, you need to find the coding sequence in the sequence supplied. To do this manually, you can look for the start site ATG and stop sites to find the coding sequence. However, there is an easier way, and this is often useful if you have more than one coding sequence from the same transcript (these might be listed as separate entries in the database): find the label "CDS" (coding sequence) under the "Features" section and click on it. It should highlight a portion of the sequence corresponding to the ORF for this coding sequence.

You can then copy this sequence directly into a the MCS of a suitable plasmid.

bob1
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