Vector for conditional knockout - (Feb/07/2009 )
I've never done knockout before, so this question might be a bit dumb
If I want to make the contruct for conditional knockout, I would need to substitute my target gene with my construct, which contain the same gene, but flanked by two loxP site (I presume)
So, there must be some commercially availble vectors which already contain the two loxP site, so that I can just easily clone my gene in there, right??
What's available in the market?
And are there particularly good vectors to do the job?
(In fact I need a second vector with the Cre-ER, but I always assume some collaborator will have the mice with Cre-ER already somewhere)
-jiro_killua-
jiro_killua on Feb 7 2009, 10:24 AM said:
I've never done knockout before, so this question might be a bit dumb
If I want to make the contruct for conditional knockout, I would need to substitute my target gene with my construct, which contain the same gene, but flanked by two loxP site (I presume)
So, there must be some commercially availble vectors which already contain the two loxP site, so that I can just easily clone my gene in there, right??
What's available in the market?
And are there particularly good vectors to do the job?
(In fact I need a second vector with the Cre-ER, but I always assume some collaborator will have the mice with Cre-ER already somewhere)
If I want to make the contruct for conditional knockout, I would need to substitute my target gene with my construct, which contain the same gene, but flanked by two loxP site (I presume)
So, there must be some commercially availble vectors which already contain the two loxP site, so that I can just easily clone my gene in there, right??
What's available in the market?
And are there particularly good vectors to do the job?
(In fact I need a second vector with the Cre-ER, but I always assume some collaborator will have the mice with Cre-ER already somewhere)
For a conditional knockout, you can use the same vector that is used for KO (Neo cassette, G418 selsection, DT/TK counter-selection otpional).
When you design your CKO strategy, you also add the LoxP site cloning. Especially if you are using recombineering, you can use the Neal Copeland system which comes with all necessary plasmids and e.coli strains free of charge from NCI. It works great.
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