6
$\begingroup$

If we insert a plasmid into a human nucleus that contains exact copy of gene and all relevant promoters to produce some human protein, will the cell create functional protein from that plasmid only when the cell needs it?

I am not talking about inserting DNA to some chromosome. Inserting separate plasmid to the nucleus (transient transfection).

$\endgroup$
8
$\begingroup$

If you transfect cells with plasmid then these plasmids need to go into the nucleus (otherwise they wouldn't be transcribed). Getting the plasmid either happens during cell division (when the nucleus is not present) or by adding a signal sequence to the plasmid which induces the import into the nucleus through nuclear pores. The SV40 sequence is an example of such a sequence.

If you have the right promoter present on the plasmid, you can basically express any protein in the cell (unless it's toxic). The promoter either needs to come from the organism from which the cells are or has to be something universally activated like the CMV promoter (from the cytomegalo virus).

A good example for this is the expression of green fluorescent protein (GFP) which originates from jellyfish and can be expressed in cells to make them glow green (from here):

enter image description here

Untransfected cells or cells transfected with an empty plasmid (without GFP) don't glow.

Targeting specific cells or control the specific activation of genes only in one cell type is possible. For both you need sequence (and cell specific) promoter sequences which either allow the nuclear import (see reference 1-3) or the expression from a cell type specific promoter (see reference 4).

This has been done in the liver of mice in which the ApoE has been knocked-out. The expression of the human ApoE gene with a specific promoter led to the correction of the hypercholesterolemia the mice were suffering from. This needs a lot of knowledge on the promoter as well as on the other transcriptional regulation elements in the specific gene (see reference 4).

References:

  1. Cell-specific nuclear import of plasmid DNA.
  2. Tissue-specific and transcription factor-mediated nuclear entry of DNA.
  3. Progress and prospects: nuclear import of nonviral vectors.
  4. Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery.
$\endgroup$
  • $\begingroup$ Thank you, Chris. I edited the question, it may be not clear, I mean some human protein, that existing in normal human cells. Lets say - the DNA that code for that protein is damaged and now we inserting plasmid with working one. So will the cell create now working protein too, and only when the cell need it? Thank you again. $\endgroup$ – Robertos Jan 30 '15 at 12:06
  • 1
    $\begingroup$ That's harder, I will edit the answer. $\endgroup$ – Chris Jan 30 '15 at 12:08
  • $\begingroup$ @chris GFP comes from Quail? I thought it came from a jelly fish. And nuclear entry isn't easy, plasmids don't just enter the nucleus. In rapidly dividing cells, they can enter when the nucleus breaks down during mitosis. Getting them to move through the nuclear pore complex is tougher. $\endgroup$ – user137 Jan 30 '15 at 14:56
  • $\begingroup$ @user137 You are right, I used the wrong word. Also about the entering of the nucleus. I edited the posting, thanks for the correction. $\endgroup$ – Chris Jan 30 '15 at 15:01
  • $\begingroup$ @Chris The SV40 sequence is an interesting one. David Dean has done a lot of work on cytoplasmic transcription factors binding to plasmid and pulling it into the nucleus. My boss doesn't believe it, I'm undecided. Quite a few papers about it, but I haven't tried any experiments myself. I suspect the transfection agent used can alter how well it works, because if your PEI or lipofectamine or whatever doesn't dissociate, the transcription factors might not be able to reach their binding sites. $\endgroup$ – user137 Jan 30 '15 at 15:40

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.