CMHD Logo Toronto Centre for Phenogenomics  
Centre for Modeling Human Disease
Home Contact Us Careers at TCP Sitemap TCP Calendar
 

Gene Trap FAQs

How do I order clones?

How do I find a gene trap clone for my gene of interest?

How do I figure out where the vector insertion has occurred within my gene of interest to make the best choice of the clones available?

Multiple clones are listed for the same gene, slightly different sequences for each, even within the same centre. Why is this?

Do all of your clones exhibit reporter gene (lacZ, gfp, venus, depending on vector) expression?

Where can I get chimeric mice made from CMHD clones?

What is the cell line used for gene trapping and where can I find out more information?

Do you do mycoplasma and pathogen testing on each individual gene trap clone?

How are the clone confirmations done and can I get a report of the results?

How should I store the ES cells when I receive them?

Can I also receive the parental R1 cells?

 Do you ship to Hong Kong?


How do I order clones?

CMHD gene trap clones are distributed by the Canadian Mouse Mutant Repository(CMMR). Information about ordering, services, and pricing can be found on their web site (http://www.cmmr.ca/services/index.html).

How do I find a gene trap clone for my gene of interest?

To search the CMHD database, please go to http://www.cmhd.ca/genetrap/database/search_sequence.html

Here you can search for your gene of interest in many different ways, gene name, Entrez gene ID, GO term, Ensembl gene ID, clone ID, blast your gene sequence etc.

There are several other ways to find a gene trap clone for your gene. The IGTC site has a blasting service available where you can enter the sequence of your gene of interest and it will search all the IGTC centres for a matching clone. This service is found at : http://www.genetrap.org. There is also a tutorial available at IGTC that helps you go through the process with all the different search engines available on their website at http://www.genetrap.org/tutorials/locate.html

You can also use Ensembl (http://www.ensembl.org/Mus_musculus/index.html) to check if there are gene traps available for your gene, although this resource might not be as up to date as IGTC, or the databases located at the individual websites for the various gene trap centres around the world, as listed below.

CMHD http://www.cmhd.ca/genetrap/database/search_sequence.html
Mammalian Functional Genomics Centre http://www.escells.ca/
BayGenomis http://baygenomics.ucsf.edu/
Exchangeable Gene Trap Clones: http://egtc.jp/view/index
German Gene trap Consortium http://tikus.gsf.de/
Sanger Institue Gene Trap Resource http://www.sanger.ac.uk/PostGenomics/genetrap/
Soriano Gene Trap Mutagenesis http://www.fhcrc.org/science/labs/soriano/trap.html
TIGEM-IRBM Gene TRAP Project http://genetrap.tigem.it/public/

How do I figure out where the vector insertion has occurred within my gene of interest to make the best choice of the clones available?

There are several different web based programs that can help you to determine this.

Here is a short tutorial on how to utilize Ensembl for this purpose.

Ensembl Viewing of Clones

One way to visualize gene trap insertions is to go to the Ensembl browser and find your gene of interest, either by blasting a sequence (http://www.ensembl.org/Multi/blastview) or looking for the gene ID in the mouse database (http://www.ensembl.org/Mus_musculus/index.html).

Once you have your gene selected, it will usually be as a GeneView window, look at the information within the genomic location section and click on the link at the end of “the start of this gene is located in “Contig …”. Once in the ContigView, you will see a section labelled Detailed view with various pull down menus located across the top. This allows you to customize the browser. Click on the DAS Sources (for "Distributed Sequence Annotation System") you will see the "Gene Trap" as one of the selections. Check this box and then select "close menu" at the bottom of the drop down menu. The window will reload with the new settings.   The gene trap clones listed at the top of the detailed view window indicate that the gene trap sequence tag maps to the sense strand of the mouse chromosome while the ones listed below the detailed view window indicate mapping to the bottom strand of the chromosome.  Make sure that your gene of interest and the gene trap sequence tags are in the same orientation. If your gene of interest and the sequence tags are located in the opposite strands of the chromosome, that means the gene trap is an anti-sense insertion. If you find one that is within your gene of interest (keep in mind that the whole length shown there represents a large part of the chromosome) you can click on the gene trap and within the menu that comes up you select DAS link: Gene trap info. This should take you to the website specific to the centre that produced the gene trap.

Multiple clones are listed for the same gene, slightly different sequences for each, even within the same centre. Why is this?

Multiple hits for the same gene happens over time, within and between centres. We have been doing gene trapping for the last 6 years and have used several different types of vectors in the process. If your gene of interest has several different gene trap clones, even from different genomic centres, it is best to take the time to look at the type of vector that was used, as well as the site of the insertion of the vector within your gene of interest. Different insertion sites and different gene trap vectors produce different alleles which can lead to different phenotypes depending on the structure of the gene or vector. The type of vector can influence such things as different reporter and selection markers, NMD mechanisms, or the possibility for cre excision to increase mutagenicity of the vector. The differences between the vectors that our group uses can be found here [WILL BE AVAILABLE SOON]

Do all of your clones exhibit reporter gene (lacZ, gfp, venus, depending on vector) expression?

Not always. Unless the reporter gene has an IRES preceding it (as in pMS1 and UPATrap) expression will only result if the insertion of the reporter gene ends up being in the correct reading frame with the gene that it was inserted into. Expression also depends on when your gene is expressed. Most gene trap vectors require expression in undifferentiated ES cells to trap the gene. The polyA trap vectors have changed this requirement and allow for unexpressed genes to also be trapped. If a trapped gene is only expressed later on in development, or in only certain types of cells, expression could be seen upon differentiation of the cells into specific lineages that do express the gene.

Where can I get chimeric mice made from CMHD clones?

CMHD gene trap clones are distributed by the Canadian Mouse Mutant Repository (CMMR). The CMMR is located at the Toronto Centre for Phenogenomics. Details on the services provided by the CMMR, including the making of chimeric mice, can be found at their web site (http://www.cmmr.ca/reference/faqs.html).

What is the cell line used for gene trapping and where can I find out more information?

We use the R1 ES cell line and more information can be found at http://www.mshri.on.ca/nagy/r1.htm

Do you do mycoplasma and pathogen testing on each individual gene trap clone?

The parental R1 cell line, MEFs , LIF and FBS lots were all tested in 2001 and we have the documentation for those. You can download an electronic version of the results by clicking here. The gene trap clones are not retested individually. In 2007, we pooled 5 clones for testing and the test results were all negative. The Canadian Mouse Mutant Repository, the distributor of CMHD clones, and the Transgenic Core and Specialty Resources, both located at the Toronto Centre for Phenogenomics, offer clone expansion and pathogen testing services. Details of these services can be found at their web site (http://www.cmmr.ca/services/index.html).

How are the clone confirmations done and can I get a report of the results?

Since the gene trapping is done in a high through-put manner and the original screen for gene insertions is done by 3’RACE, we also isolate RNA from the thawed clone and perform RT-PCR to confirm each clone request. We do this by taking an aliquot of cells at the time of expansion and culturing them on gelatine for at least one passage. We then isolate RNA and perform RT-PCR, using random hexamers to produce cDNA, followed by a PCR with a primer specific to the 3’ end of our vector (usually in the neo gene) and another one which is gene specific downstream of the vector insertion site (designed by our technicians when you order your clone). The resulting product is sent for sequencing to confirm that the sequence following the vector is indeed your gene of interest. We are in the process of generating an electronic version of these results, including the PCR primers used as well as the sequencing results and trace files, which will be posted on our website under the specific clone IDs. This process and protocol used to confirm your clone is available in the “How to” section. Trace files received from the sequencing of expanded clones will be added to the individual clone report on our website which you can download and view in a program such as BioEdit (free on the internet). Until we have the electronic version available on our website, you can request that the PCR and sequencing results be sent to you. Please email to request this information.

How should I store the ES cells when I receive them?

Two vials of your clone will be shipped to you on dry ice and you should place your vials of cells directly into LN upon receiving them. They can be temporarily stored at -80 but we can not guarantee the quality of the cells upon thawing if they have been stored at -80 for any length of time.

Can I also receive the parental R1 cells?

The R1 parental line of ES cells that were used for the generation of the gene trap clones should be requested from the Nagy lab at Mount Sinai Hospital http://www.mshri.on.ca/nagy/r1.htm .

Do you ship to Hong Kong?

FedEx will not ship any package that contains dry ice to Hong Kong, due to restrictions at the Hong Kong end of things unless special arrangements are made. Arrangements are made by the requester for shipments to be delivered to a FedEx depot within Hong Kong where the package can be released and picked up by the requester. Please look at the FedEx website for Hong Kong restrictions. http://www.fedex.com/us/international/irc/profiles/irc_hk_profile.html?gtmcc=us#C04

Back to Top

Mount Sinai Hospital
CMHD Logo