how to generate the target construct ?

[ruby]

I am new to this field and have good molecular biology background but never made a KO.

I have been given a task of making a KO of a particular protein. I know the accession no. of it cDNA. Its in a pET vector.

How to go about it ? what all should i read and gather information in order to make th econstruct, keeping in mind the screening strategy of the clones in furture.

Any help/suggestion/comments are welcome,
thanks,
ruby

[kmunson779]

Try this site (scroll down to the grey additional information box).

ETA: actually, this one looks like more what you want.

[ruby]

thanks kmunson779,

I already have these links, i think i should ask u specific questions, the reason, i didnot do it initially as these r very BASIC questions, infact some of them are trivial too, anyway I will give a try. I will ask few at a time:

1) i have the information about the cDNA clone, how to get the information about genomic clone. I am not sure if there is a genomic library available commercially, which can be screened. how to look for enhancers or any other regulatory sequence ?

2) how to get the accession no. of a genomic clone ? which DNA segment to consider as long arm and which one as short arm ?

3) i got the gene sequence (NCBI) from murine source, how much homologous is it with its respective human gene, i am not sure?

Thanks for your help.
ruby

[kmunson779]

I will predicate this post with the disclaimer that I have never done this myself (I'm a fly person... we just got the technology to consider this a few years ago).

1) i have the information about the cDNA clone, how to get the information about genomic clone. I am not sure if there is a genomic library available commercially, which can be screened.

This is in that second link above (under Step 2). Shortly, look up your gene in the mouse database at ensembl.org.

how to look for enhancers or any other regulatory sequence ?

This is trickier... Search PubMed for papers on your gene is the first answer; other than that people do bioinformatic analysis of the upstream sequence to look for transcription factor binding sites, and biochemically, deletion analysis of that sequence. I'm not sure how this is relevant to creating a knockout, though.

2) how to get the accession no. of a genomic clone ?

Ensembl.org or one of the other sites mentioned in step 2.

which DNA segment to consider as long arm and which one as short arm ?

Step 5, and look for good restriction sites; try to get a "long" fragment and a "short" one.

3) i got the gene sequence (NCBI) from murine source, how much homologous is it with its respective human gene, i am not sure?

I use homologene for these sorts of questions. Ensembl has a similar tool (under Alignments, choose "view genomic alignment with Homo sapiens"). This might also be in the literature, or you can download the respective sequences and align them yourself with clustalw or the like.

[ruby]

thanks kmunson 779,

thanks for the help, I will try these suggestions. I will get back for further clarification,

ruby

[krisztina]

I also thank for the informations and for the links. I have the same task> generating KO mice though I never did it before.

[knockout maker]

Hi there,
We are doing knockouts as a internal facility service so I guess it would be interesting for you to know how do we proceed:
First of all you should get familiar with the ensembl database and molecular biology softwares like vector NTI (which you can download for free) or Lasergene. Secondly, it is best to order a BAC clone containing you genomic insert instead of screening lambda phages or BAC libraries by classic filter hybridization or PCR. You can easily order your genomic clones containing your insert of interest from BacPac resources at CHORI (for B6 clones) or at the sanger (they do not let me post websites addresses in this forum yet otherwise there would be many in this reply) for a 129 background BAC clone. Once you have those you can follow a three steps recombineering protocol as Pentao Liu suggest in his last paper (A recombineering based approach for high-throughput conditional knockout targeting vector construction) or just subclone a genomic piece from the BAC directly by restriction digest and clasical ligation and transformation. For that you should build up a complete map of your insert in the BAC backbone (do not forget to add the BAC backbone sequence) and check whether a restriction digest containing your gene of interest is suitable at all. We have good experiences doing that and some other people too (Local and cis effects of the H element on expression of odorant receptor genes in mouse). A third strategy would be to amplify two big DNA pieces (2-5Kb) from the BAC (easier than fro genomic DNA) as homologies for subsequent gene targeting and subclone those sequentially in a customized targeting vector such as pFLEX. Depending on the strategy you want to follow and you specific question you will make constitutive, conditional or knock-in constructs.
For the screening...lastly pleople are using long-range PCR, meaning using an external primer to your knockout construct and directly amplifying from the ES cells clones with a complementary primer in the selectable cassette. If you find a positive then you can proceed to do a southern of a couple of clones instead of doing a southern for every clone. Designing a Southern is not trivial and molecular biology software should be used. In general we like to have the putative strategy worked out before the cloning started, so on can plan in advance what type of restriction sites should be introduced during the cloning to make the Southern easier.
I would advice you first click around in ensembl, get the vector NTI from invitrogen, order your BAC, request recombineering reagents from the recombineering website (type recombineering in google) and get ready for what is to come. You can also contact the american knockout mouse project or the european conditional mutagenesis program and ask them to prioritize your gene so that they generate it for you for free...
Good luck!!!

[knockout maker]

Hi there,
We are doing knockouts as a internal facility service so I guess it would be interesting for you to know how do we proceed:
First of all you should get familiar with the ensembl database and molecular biology softwares like vector NTI (which you can download for free) or Lasergene. Secondly, it is best to order a BAC clone containing you genomic insert instead of screening lambda phages or BAC libraries by classic filter hybridization or PCR. You can easily order your genomic clones containing your insert of interest from BacPac resources at CHORI (for B6 clones) or at the sanger (they do not let me post websites addresses in this forum yet otherwise there would be many in this reply) for a 129 background BAC clone. Once you have those you can follow a three steps recombineering protocol as Pentao Liu suggest in his last paper (A recombineering based approach for high-throughput conditional knockout targeting vector construction) or just subclone a genomic piece from the BAC directly by restriction digest and clasical ligation and transformation. For that you should build up a complete map of your insert in the BAC backbone (do not forget to add the BAC backbone sequence) and check whether a restriction digest containing your gene of interest is suitable at all. We have good experiences doing that and some other people too (Local and cis effects of the H element on expression of odorant receptor genes in mouse). A third strategy would be to amplify two big DNA pieces (2-5Kb) from the BAC (easier than fro genomic DNA) as homologies for subsequent gene targeting and subclone those sequentially in a customized targeting vector such as pFLEX. Depending on the strategy you want to follow and you specific question you will make constitutive, conditional or knock-in constructs.
For the screening...lastly pleople are using long-range PCR, meaning using an external primer to your knockout construct and directly amplifying from the ES cells clones with a complementary primer in the selectable cassette. If you find a positive then you can proceed to do a southern of a couple of clones instead of doing a southern for every clone. Designing a Southern is not trivial and molecular biology software should be used. In general we like to have the putative strategy worked out before the cloning started, so on can plan in advance what type of restriction sites should be introduced during the cloning to make the Southern easier.
I would advice you first click around in ensembl, get the vector NTI from invitrogen, order your BAC, request recombineering reagents from the recombineering website (type recombineering in google) and get ready for what is to come. You can also contact the american knockout mouse project or the european conditional mutagenesis program and ask them to prioritize your gene so that they generate it for you for free...
Good luck!!!