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Mouse Embryonic Stem (mES) Cells

Service Details

Service Charges

How To Order


Service Details

NorCOMM Gene Trap mES Cell Lines

The CMMR is the repository and distribution centre for the NorCOMM gene trap mES cell library. Before ordering your gene trap lines, we strongly recommend that you carefully review the data available for the CMHD and ESDB gene trap databases and blast the gene trap sequence tag against the Ensembl mouse genome, USCS genome browser and/or Ensembl mouse genome to make sure that the sequence tag maps to your gene of interest and that it is in the sense orientation with the gene. It is also important to review the vector information to make the best choice among available lines. Upon receipt of a request, the bioinformatics associated with the requested line is reviewed to see if any obvious caveats are associated with a given line. If a problem upon bioinformatics review is detected, the Requester is contacted with the information and asked whether s/he would like to confirm or rescind the order. If no problems are detected, the Request is approved and put in the queue for expansion.

For each Gene Trap mES cell line order, lines are bioinformatically reviewed, thawed, expanded, frozen into aliquots and sequence validated by either 3'-RACE, RT-PCR, splinkerette PCR, inverse PCR or genomic PCR and sequencing of the resultant PCR product. SOPs associated with the procedures performed at CMMR can be found on our SOP website. Sequence confirmation of mES cell lines may be done at the CMMR or at the production facility (MFGC or CMHD). After sequence confirmation, two cryovials containing the expanded line are shipped on dry ice to the Requester at his/her expense. Requests normally take 8-12 weeks for expansion and validation before lines are shipped.

Not-for-profit investigators/institutions should carefully read and make sure they understand the CMMR Undertakings of Use. In order for their request to be processed, not-for-profit investigators/institutions must agree to abide by these terms.

For-profit investigators/institutions must execute a Material Transfer Agreement (MTA) with the mES cell production facility (University of Toronot for CMHD mES cell lines and University of Manitoba for MFGC mES cell lines). The CMMR will provide the appropriate template to the Requester. Upon return of two original signed copies of the MTA to the CMMR, the request will be put in the queue for expansion.

Service fees for Gene Trap mES Cell Lines include bioinformatics review, line thawing, expansion, freezing and sequence validation. Service fees do not include shipping charges, including anyc costs to return dry shippers to the CMMR. Service fees must be paid in full by cheque or credit card, or by receipt of a valid institutional purchase order, before cells are shipped to the requester.

NorCOMM Targeted mES Cell Lines

The CMMR is the repository and distribution centre for the NorCOMM targeted mES cell library. As part of the International Knockout Mouse Consortium (IKMC), NorCOMM targeted cell lines are in the B6N-derived C2 cell line. The NorCOMM targeting cassette results in a null allele that replaces a critical region (comprised of one or more exons) with a E. coli lacZ reporter construct (Figure 1). Among other criteria, deleted exons are selected based on their presence in all annotated transcripts of the gene and their removal introduces a frame-shift in the coding region. During production and screening, targeting is confirmed by long PCR with primers beyond the end of the 5' homology arm and within the targeting cassette.

Figure 1. Shematic of the NorCOMM Targeted Trap Allele.

For each Targeted mES Cell Line order, lines are thawed, expanded, frozen into aliquots and genoytped by long PCR across the 5' homology arm junction (Figure 2). PCR products are end sequenced to confirm genomic location. However we strongly recommend that users confirm targeting by Southern blot analysis or by carefully designed quantitative PCR prior to using cell lines to generate mice. SOPs associated with the procedures performed at CMMR can be found on our SOP website. After sequence confirmation, one cryovial containing the expanded line is shipped on dry ice to the Requester at his/her expense. Requests normally take 8-12 weeks for expansion and validation before lines are shipped.

Figure 2. Schematic of NorCOMM allele targeting confirmation for targeted mES cell lines.

Not-for-profit investigators/institutions should carefully read and make sure they understand the CMMR Undertakings of Use. In order for their request to be processed, not-for-profit investigators/institutions must agree to abide by these terms.

For-profit investigators/institutions must execute a Material Transfer Agreement (MTA) with the mES cell production facility (University of Toronto for CMHD mES cell lines and University of Manitoba for MFGC mES cell lines). The CMMR will provide the appropriate template to the Requester. Upon return of two original signed copies of the MTA to the CMMR, the request will be put in the queue for expansion.

Service fees for Targeted mES Cell Lines include line thawing, expansion, freezing and sequence validation. Service fees do not include shipping charges, including any costs to return dry shippers to the CMMR. Service fees must be paid in full by cheque or credit card, or by receipt of a valid institutional purchase order, before cells are shipped to the requester.

NorCOMM mES Cell Line Antibiotic-Free Passage

Due to the high-throughput nature of NorCOMM mES cell line production and medium-throughput nature of mES cell line distribution, pathogen testing is done in batches rather than testing of individual lines (see our pathogen testing policy for more information). While the CMMR is an approved mES cell facility for the generation of chimeric mice at the Toronto Centre for Phenogenomics Transgenic Core, eliminating the need for additional testing; other facilities may require that cells be tested for common murine pathogens prior to the generation of chimeric mice. Testing often needs to be preceded by antibiotic-free passage (AFP) of cells. The CMMR will produce AFP cell aliquots for a nominal fee in parallel with NorCOMM mES cell withdrawal when requested at the same time.

For each line, Antibiotic-Free Passage, involves three passages of mES cells without feeders and without antibiotics. Two vials of AFP cells are then supplied to the investigator with their expanded lines of interest as frozen cells in media or PBS, as requested. AFP ES cells are not suitable for experiments other than pathogen testing.

Service fees for Antibiotic-Free Passage include passage and freezing of cells. Service fees do not include shipping charges.

Generating Chimeric Mice from ES Cells

Through the TCP Transgenic Core, investigators can request attempts to generate chimeric mice from mES cells. These requests are normally preceded by NorCOMM mES Cell Withdrawal or Expansion of ES Cells Clones and Their Preparation for the Generation of Chimeras. When requested with an order for NorCOMM ES Cell Withdrawal, expanded mES cell lines can be thawed and transferred from the CMMR to the Transgenic Core for a nominal service fee. Through the CMMR, investigators can recover mice from mES cells supplied by FIMRe member or other repositories. Details regarding the generation of chimeric mice from mES cells at the TCP can be found at Transgenic Core: Generation of Chimeras. Please contact or for more information.

NorCOMM Targeting Vectors

All targeting vectors produced by NorCOMM are available from the CMMR. Upon receipt of an order, the vectors are purified, end sequenced, and restriction mapped with one or two restriction endonucleases. Vector is spotted onto filter paper and shipped dry to Requesters via regular mail. Derived vector sequences are emailed to the Requester upon completion of the request.

Service Charges (Canadian Funds), as of April 1, 2010

Service TCP Investigators Non-Profit Canadian Institution Non-Profit International Institution
NorCOMM Gene Trap mES Cell Lines, per line
  • Includes bioinformatic review, line thawing, expansion, freezing and sequence validation
  • Does not include shipping

$500

$500

$750

NorCOMM Targeted mES Cell Lines, per line
  • Includes bioinformatic review, line thawing, expansion, freezing and sequence validation
  • Does not include shipping

$650

$650

$650

NorCOMM mES Cell Line Antibiotic-Free Passage, per line
  • Includes three passages of expanded line in antibiotic-free media and subsequent freezing of cells in vials for pathogen testing
  • Available only in conjunction with NorCOMM ES Cell Line Withdrawal
  • Does not include shipping or mouse pathogen testing

$100

$130

$160

Additional Vial of NorCOMM mES Cells, per vial
  • Includes expansion and freezing of additional vials of ES cells
  • Available only in conjunction with NorCOMM ES Cell Line Withdrawal
  • Does not include shipping

$160

$160

$256

NorCOMM Targeting Vector, per vector
  • Includes purification, end sequencing and restriction mapping of target vectors
  • Delivered dry by regular mail

$260

$260

$320


How to Order

The Principal Investigator (PI) (and designate) must have an account on TCP LIMS. Please follow the Order Instructions for ES Cells.

 

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