How complementation works - basic question (Apr/12/2009 )

Need help understanding some basic aspects of complementation genetics.

Say I have a WT gene transcript, where this particular gene encodes for a particular protein. Now I knock out the expression of the gene so that it can't make the protein (knockout mutant). I do this by making mutations on the gene so that the expression of the protein will not be made.

Now, I am still not 100& sure that that mutation caused the knockout or if I disrupted something else in the mechanism that also caused the knockout. So I want to create a complement line to test if the knockout is really linked to the mutation.

This is where I am stuck: To make a complement line, how exactly do you go about this? Can you just take the wild-type gene sequence of that protein and place it back into the knockout gene transcript? Or can you 'fix' the mutations? And how do you go about doing that?

Hey,

Don't know a lot abt this but this is what I think they do....You put yr wild type gene in another plamid that will express in yr organism under study. Now you electroporate this plasmid in the mutant strain and study the effect, if it reverses then teh observed phenotype you see in teh mutant is because of the target gene.

Hope it helps,

Best,
TC

claritylight on Apr 13 2009, 04:08 AM said:

T C is right!

You simply express your wildtype gene on a plasmid which is transformed into your mutant.
As a control you also need the "empty" plasmid in your mutant strain, to make sure that the plasmid itself doesn't effect your strain.