Knock out mouse construct-plz help - (Aug/26/2008 )
Dear All
I am at present learning on gene knock-out technology. To make a targeting vector for knock out of a particular gene is there any good text book, papers etc?
I have downloaded several papers but they are not talking in detail as to How To Make The Construct.So far I know through homologous recombination an engineered constuct could be inserted into the mouse genome and then by cre/lox recombination particular gene of interest could be deleted.
I want to know each steps from the begining i.e. how to make the targeting vector that will disrupt the targetting alelle etc.
Please help me. :
thx in advance.
( I made several corrections of my former message , sorry)
I am currently doing a knockout myself and didn't come across any good book but I can try give you some information for a start:
1) Plan the strategy
Usually you design a construct containing a selectable marker that integrates site-specifically and thereby it will disrupt your gene of interest. You need to define where you want it to integrate and what selectable marker to use.
Where?
I am targeting the start codon and I will also try to delete a splicing signal and mess up the reading frame so that no (functional) protein can be produced.
Marker?
I use a resistance gene for my cell line, I don't know what people ususally do in mice. If you flank your disruption cassette by loxP sites you can delete the cassette after successful targeting by Cre recombination, which is quite handy. Make sure the gene is still non-functional after Cre-mediated excision of the cassette. Also think of a way to screen for gene targeting by Southern Blotting (include some additional restriction sites for example)
2) Cloning
Once you have developed your strategy you can start cloning the targting vecor. You need two arms of homology targeting the construct to the locus, the longer the better, mismatches greatly reduce targeting frequency, mine are roughly 5kb each. You can simply clone these from genomic DNA (using a very good polymerase) or libraries should this not work. You can use any cloning vector as backbone for your strategy, I use pBlueskript.
3) Targeting
Before you target you linearise the construct, as close to one arm of homology as possible and transfect it into your cells, isolate cells that express your selectable marker and screen them for targeted / random integration. This should be done by Southern but can be made a little easier by designing a clever colony PCR (one primer annealing to your disruption cassette, one outside your region of homology). In cell lines we screen about 250 clones to find one with a successful knockout.
It's a long way to go :-)
Some papers I find useful (search for accession numbers in PubMed):
loxP sites: 9016639
sequence replacement vectors in ES: 1620105
considerations for the regions of homology:2854196
Good luck!!!
Thx Genny_London for ur in detail information.
It will help me a lot. Ok, I will search the papers to know further.
best regards,
Hasina
1) Plan the strategy
Usually you design a construct containing a selectable marker that integrates site-specifically and thereby it will disrupt your gene of interest. You need to define where you want it to integrate and what selectable marker to use.
Where?
I am targeting the start codon and I will also try to delete a splicing signal and mess up the reading frame so that no (functional) protein can be produced.
Marker?
I use a resistance gene for my cell line, I don't know what people ususally do in mice. If you flank your disruption cassette by loxP sites you can delete the cassette after successful targeting by Cre recombination, which is quite handy. Make sure the gene is still non-functional after Cre-mediated excision of the cassette. Also think of a way to screen for gene targeting by Southern Blotting (include some additional restriction sites for example)
2) Cloning
Once you have developed your strategy you can start cloning the targting vecor. You need two arms of homology targeting the construct to the locus, the longer the better, mismatches greatly reduce targeting frequency, mine are roughly 5kb each. You can simply clone these from genomic DNA (using a very good polymerase) or libraries should this not work. You can use any cloning vector as backbone for your strategy, I use pBlueskript.
3) Targeting
Before you target you linearise the construct, as close to one arm of homology as possible and transfect it into your cells, isolate cells that express your selectable marker and screen them for targeted / random integration. This should be done by Southern but can be made a little easier by designing a clever colony PCR (one primer annealing to your disruption cassette, one outside your region of homology). In cell lines we screen about 250 clones to find one with a successful knockout.
It's a long way to go :-)
Some papers I find useful (search for accession numbers in PubMed):
loxP sites: 9016639
sequence replacement vectors in ES: 1620105
considerations for the regions of homology:2854196
Good luck!!!
I am at present learning on gene knock-out technology. To make a targeting vector for knock out of a particular gene is there any good text book, papers etc?
I have downloaded several papers but they are not talking in detail as to How To Make The Construct.So far I know through homologous recombination an engineered constuct could be inserted into the mouse genome and then by cre/lox recombination particular gene of interest could be deleted.
I want to know each steps from the begining i.e. how to make the targeting vector that will disrupt the targetting alelle etc.
Please help me. :
thx in advance.
( I made several corrections of my former message , sorry)
Hi,
I hope this book may help
Gene Targeting: A Practical Approach
by Alexandra L. Joyner
Have a look http://www.amazon.com/Gene-Targeting-Pract...r/dp/019963792X
dear MolBio79
thx very much. i am now checking the book.
regards,
hasina
I am at present learning on gene knock-out technology. To make a targeting vector for knock out of a particular gene is there any good text book, papers etc?
I have downloaded several papers but they are not talking in detail as to How To Make The Construct.So far I know through homologous recombination an engineered constuct could be inserted into the mouse genome and then by cre/lox recombination particular gene of interest could be deleted.
I want to know each steps from the begining i.e. how to make the targeting vector that will disrupt the targetting alelle etc.
Please help me. :
thx in advance.
( I made several corrections of my former message , sorry)
Hi,
I hope this book may help
Gene Targeting: A Practical Approach
by Alexandra L. Joyner
Have a look http://www.amazon.com/Gene-Targeting-Pract...r/dp/019963792X