CMMR-NorCOMM Frequently Asked Questions (FAQs)
What is the CMMR?
What is NorCOMM?
How do I find a mutant ES cell clone for my gene of interest?
How do I order ES cell clones?
What information do I need to order clones?
What is the clone retrieval process?
Why is sequence confirmation of my gene trap clone necessary?
Where can I get more information about choosing an ES cell clone?
What is the parental cell line used for gene trapping?
How should I store the ES cells when I receive them?
How do I maintain my ES cell clones?
Do you test individual clones for mycoplasma or other pathogens?
How long will it take for me to receive my ES cell clone(s)?
Who can order ES cell clones?
How much does an ES cell clone cost?
How do you calculate your charges?
Will the CMMR recover live mice from my ES cell clone of interest?
How much does mouse recovery from mutant ES cells cost?
How do I design a genotyping protocol for mice made from mutant ES cells?
What is the NorCOMM toolbox?
How can I order vectors from the NorCOMM toolbox?
Where can I find protocols for use of the NorCOMM toolbox?
I've altered my ES cell clone(s). Can the CMMR derive mice from my modified ES cell clone?
What is FIMRe?
What is IGTC?
What is IMSR?
More questions?
What is the CMMR?
The Canadian Mouse Mutant Repository, CMMR, is a facility dedicated to the preservation and distribution of mutant mouse lines, samples, and ES cells.
What is NorCOMM?
The North American Conditional Mouse Mutagenesis (NorCOMM) project is
the Canadian contribution to the International Knockout Mouse Consortium (IKMC), a large-scale international research initiative focused on developing and distributing a library of mouse embryonic stem (ES) cell lines carrying single conditional knockout mutations across the mouse genome. Two production facilities are associated with NorCOMM the Centre for Modeling Human Disease (CMHD) Gene Trap laboratory at the University of Toronto and the Mammalian Functional Genomics Centre at the Manitoba Institute of Cell Biology.
How do I find a mutant ES cell clone for my gene of interest?
Each production centre maintains a web accessible database of their ES cell clones. Clones can be searched by clone ID, gene name, gene symbol, Entrez, Unigene, MGI, or Ensembl gene IDs, InterPro ID, GO terms, or other keywords, depending on the production facility database. Databases enable BLAST searching of their ES cell clone sequence tags. Once you’ve found your clone of interest, make a note of the complete clone ID, MGI gene symbol of the trapped/targeted gene and the production facility (CMHD or MFGC). You can order NorCOMM clones from the CMMR online (see How do I order ES cell clones? below).
Through FIMRe, the CMMR can recover live mice from ES cell clones produced by the IGTC and others and held at FIMRe member repositories. Import of ES cells from international repositories is often faster and subject to fewer restrictions than the recovery of live mice. The IGTC resources can be searched at the IGTC website. Comprehensive searches of ES cell clones and live and cryopreserved mutant mouse strains can be done at the IMSR website.
Sources of mutant ES cells include:
The production facility websites have valuable information about the availability of different clones, gene trap or targeting vectors used, parental cell line(s) used, reporter gene expression (if any) and other characteristics and considerations about the ES cell clones they have available.
How do I order ES cell clones?
For NorCOMM ES cell clones held at the CMMR, place an order through our online Laboratory Information Management System (LIMS). New users must first register for a user account. Your Principal Investigator (PI) must also have a registered account on LIMS.
- Assemble all information needed to place your order (more details…).
- Register and log in to TCP LIMS .
- From the Service Requests menu, select "Request a TCP Service".
- Search for Department "Canadian Mouse Mutant Repository (CMMR)".
- Select the appropriate withdrawal form from the list of available services:
- Investigators from not-for-profit institutions select ES Cell Clone Withdrawal, Not-for-profit
- Investigators from for-profit institutions select ES Cell Clone Withdrawal, For-profit
- Click the magnifying glass next to Principal Investigator and select the appropriate PI name.
- Fill in the remaining information on the Contact Information and User Forms using the information gathered in Step 1.
Investigators from not-for-profit institutions must read and accept the Terms and Conditions of Use. Investigators from for-profit institutions must execute two original copies of the appropriate MTA. In the latter case, an electronic form copy of the MTA will be forwarded to you by email once your request has been accepted and bioinformatics review completed.
- Submit your request.
For non-NorCOMM ES cell clones (ie. those from international repositories), please contact us
What information do I need to order clones?
Service Request forms cannot be saved or edited after submission. Please make sure you have all the following information before placing your order:
- Principal Investigator name (principal investigator must be previously registered in the TCP LIMS)
- Contact (PI or Designate) name, telephone, fax, email, and mailing address
- Billing information with payment method:
- TCP Member Hospital Investigators: Cost centre to which your requests should be billed
- Other investigators: Preferred payment method and purchase order number, if necessary
- Complete shipping information:
- Shipping address with room and/or floor number, building, street address, city, (province/state), country and postal/zip code. Note that couriers will not normally deliver to postal box addresses. If additional room is needed, use the comments section on the request form.
- Courier and account information should be specific for international shipments, if ordering from outside of Canada. All cells will be shipped packaged in dry ice, so be sure that the courier you use accepts these types of shipments. Our customers commonly use DHL, FedEx and World Couriers.
- Courier name and account number.
- Complete clone ID (from CMHD, MFGC, IGTC, IMSR or other database), MGI gene symbol of the trapped or targeted gene, and the name of the production facility (CMHD or MFGC) or supplying repository (for non-CMMR stock clones).
- For ES cell clones, service charges must be paid in full prior to cells being shipped. This can be done by credit card, bank transfer, cheque, or money order, or by a valid institutional purchase order to the CMMR (use “The Hospital for Sick Children” as the vendor name).
What is the clone retrieval process?
Upon receipt of a request for a NorCOMM clone, the production facility bioinformatician reviews the clone request and the data associated with the requested clone. If necessary, a summary of the information for a particular clone and request for confirmation of the order will be emailed to the Requester. Once the Requester confirms their order or if no problems are identified during bioinformatics review, the clone retrieval process is initiated at the CMMR. This process involves the following steps:
- The clone is thawed and expanded per SOPs for the parental cell line.
- Once expanded, five aliquots of ES cells are frozen and stored and one aliquot is transferred to the originating production facility for nucleic acid isolation and sequence tag confirmation.
- Once the gene trap sequence is confirmed, two aliquots of frozen ES cells are shipped to the Requester on dry ice or in a cryo-shipper by overnight express courier at the Requester’s expense.
- Clone information and sequence confirmation reports are included with the ES cell clone shipment or requests for electronic copies of this documentation can be made by email to the CMMR
Why is sequence confirmation of my gene trap clone necessary?
High-throughput gene trapping and sequence tag acquisition necessarily rely on universal primers for either genomic DNA PCR or 5’- or 3’-RACE-PCR for sequence tag acquisition. To confirm the gene trapped sequence, we use either iPCR or gene-specific primers (GSPs) from the original tagged sequence for genomic DNA PCR or RT-PCR. The PCR product is sequenced to confirm that the originally identified gene is trapped in that ES cell clone.
Where can I get more information about choosing an ES cell clone?
When multiple clones targeting a single gene exist, the selection of the clone depends on the needs of the end user. Suitability of the clone is dependent on the site of insertion, the gene trap vector used, and in some cases, the genetic background of the parental ES cell line. The CMHD How To and FAQs websites offer information on their vectors and how to choose a mutant ES cell clone
from their library. The MFGC also has an FAQ section on their website. .
What is the parental cell line used for gene trapping?
Information on parental cell lines used for gene trapping can be found at the production facility websites and/or in their ES cell clone databases. It is essential to follow the SOPs for ES cell propagation specific to the parental cell line used to make your gene trap in order to preserve totipotency.
How should I store the ES cells when I receive them?
ES cell aliquots should be stored in liquid nitrogen immediately upon receipt.
How do I maintain my ES cell clones?
Information on parental cell lines used for gene trapping can be found at the production facility websites. It is essential to follow the SOPs for ES cell propagation specific to the parental cell line used to make your gene trap in order to preserve totipotency. The SOPs used by the CMMR for propagation of NorCOMM ES cells can be found on our website.
Do you test individual clones for mycoplasma or other pathogens?
Due to the high throughput nature of our trapping and targeting pipelines, it is neither practical nor cost effective to test each clone. ES cell clones are produced in closed, designated ES cell facilities following strict protocols to ensure cleanliness of the facility and clones. Each NorCOMM production facility routinely tests their parental ES cell lines and their feeder cell lots for mycoplasma and other mouse pathogens (see NorCOMM SOPs).
At the CMMR, we randomly select three to five clones for mouse pathogen and mycoplasma testing each quarter (IMPACT TCP Profile IV testing at RADIL). All our feeder cell lots are mouse pathogen and mycoplasma tested before use (RADIL IMPACT Profile I). ES cells are grown in a closed, designated mouse ES cell facility following strict protocols to ensure cleanliness of the facility and clones. Please see our SOP ESC03, “Quarterly pathogen testing of embryonic stem cell clones”.
How long will it take for me to receive my ES cell clone(s)?
After bioinformatics review, ES cell clone requests are put into the distribution queue. When bioinformatics review identifies potential problems, the Requester is notified and asked to confirm, change, or rescind the order. Where bioinformatics review has uncovered a possible concern, clone recovery is not initiated until order confirmation is received. Orders from for-profit institutions will not be processed until two signed original copies of the relevant MTA are received from the Requester. Once started, clone recovery, including sequence confirmation, takes 6-8 weeks. The start date for clone recovery depends on the number of requests and clones in the queue at the time of order confirmation.
Who can order ES cell clones?
Generally, an investigator associated with a recognized academic research institution or biomedical/biopharmaceutical company can order ES cell clones.
How much does an ES cell clone cost?
Our services and fees are detailed on our website. ES cell clones are shipped to the Requester at his/her expense with the provision of a courier account number at the time of IMS User Registration. Service charges for ES cells must be paid in full by credit card, cheque or bank transfer, or by a valid institutional purchase order to the CMMR (use”The Hospital for Sick Children” as the vendor), prior to cells being shipped.
How do you calculate your charges?
FIMRe member repositories are attempting to harmonize charges world-wide so that geographical location is not a limiting factor in accessing the world’s mouse mutant resources. The CMMR includes the cost of sequence validation in its fees, in contrast to some other repositories.
Fees charged to Canadian not-for-profit investigators are subsidized by operating grants to NorCOMM facilities. Therefore, charges to Canadian investigators are somewhat less than those charged to international requesters.
Will the CMMR recover live mice from my ES cell clone of interest?
The CMMR and TCP Transgenic Core and Specialty Resources work together to generate chimeric mice from ES cell clones. Generation of chimeric mice involves expansion of the ES cell clone, mouse pathogen (IMPACT) testing, if necessary, embryo aggregation or blastocyst injection, and embryo transfer. Once litters have been born, mating to assess germline transmission can be done at the TCP or chimeras can be shipped directly to the Requester at their home facility. To generate chimeric mice from CMMR stock ES cells, the Requester must first place a request for ES cell clone Withdrawal and then a request for Generation of Chimeras. Please contact us for more information.
How much does mouse recovery from mutant ES cells cost?
In addition to the costs of clone recovery, costs to recover mutant mice from ES cells vary according to the procedure performed, number of chimeric mice produced, and age of mice at time of shipping to the Requester. Details of
services and fees are on our web page and for Generation of Chimeras can be found elsewhere.
How do I design a genotyping protocol for mice made from mutant ES cells?
The CMMR validates sequence tags by either RT-PCR, 3’-RACE, inverse PCR (iPCR) or splinkerette (SPLK) PCR. Protocols for these can be found with our other SOPs. The CMHD Gene Trap Core has a very good description of the steps involved in designing genotyping protocols for clones whose sequence tag was generated using 3’-RACE on their How To web page.
For gene trap clones with genomic DNA tags (iPCR or SPLK PCR), gene-specific primers should be designed to amplify a region spanning the gene trap vector insertion site. This PCR will detect the wild-type allele of the gene. The 5’ or 3’ gene-specific primer can be used in conjunction with a vector-specific primer to amplify the gene-trap allele of the gene. Vector sequences and tested vector-specific primer sequences can be found at the CMHD or MFGC website. A list of primers used by the CMMR can be found with the CMMR SOPs.
What is the NorCOMM toolbox?
The NorCOMM toolbox is a series for cloning vectors that enable the replacement of the NorCOMM allele (trapped or targeted) with other sequences. Subsequent resolution of the replaced allele using Flp recombinase removes extraneous DNA sequences leaving only the replacer sequences flanked by FRT sites at the original site of gene insertion. Details of this strategy can be found at the NorCOMM website.
How can I order vectors from the NorCOMM toolbox?
Coming soon.
Where can I find protocols for use of the NorCOMM toolbox?
Coming soon.
I’ve altered my ES cell clone(s). Can the CMMR derive mice from my modified ES cell clone ?
To generate chimeric mice from ES cells cultured and/or manipulated at an Investigator’s lab, the clone must be submitted to the CMMR (or Transgenic Core and Specialty Resources) for clone expansion and mouse pathogen (IMPACT) testing before generation of chimeras at the TCP Transgenic Core and Specialty Resources will be attempted. Details of the
ES cell clone expansion service and fees can be found on our Services and Fees pages. Mouse pathogen (IMPACT) testing is necessary to protect our clean ES cell and animal facilities from contamination. Additionally, infected ES cells perform very poorly in both aggregation and blastocyst injection and identification of these potential problems can assist the Investigator in making informed decisions. In addition to pathogen testing, the Investigator may choose to have the genomic integrity of their ES cell clone assessed either by simple chromosome counting or by full mouse karyotyping (for additional fees). Genomic assessment is optional, but may assist an Investigator in choosing between multiple clones for the generation of chimeric mice.
What is FIMRe?
The Federation of International Mouse Repositories (FIMRe) is a collaborating group of Mouse Repository and Resource Centres worldwide whose collective goal is to archive and provide strains of mice as cryopreserved embryos and gametes, ES cell lines, and live breeding stock to the research community. The CMMR is an active member of FIMRe. As such, the CMMR can assist an Investigator in the recovery of live mice from frozen stocks held at other repositories. Please contact us for more information.
What is IGTC?
International Gene Trap Consortium (IGTC) membership includes laboratories around the world working together to generate a public library of mutated murine ES cell lines. Such cell lines can be obtained on a non-collaborative basis by scientists interested in generating reporter-tagged, loss-of-function mutations in mice from associated mouse mutant repositories. In addition to loss of function, newer gene trap vectors offer a variety of post-insertional modification strategies to allow for the generation of other experimental alleles.
What is IMSR?
The International Mouse Strain Resource (IMSR) is a searchable online database of mouse strains, stocks and mutant ES cells available worldwide. Mouse strains and stocks include inbred, mutant, and genetically engineered mice. The goal of the IMSR is to assist the international scientific community in locating and obtaining mouse resources for research.
More questions?
Do you still have questions? If so, please contact us.
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