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ES Cell Services


Gene Targeting

Service Details      Service Charges      How To Order


Service Details

We will perform electroporation of the targeting construct, isolate individual clones that have survived the selection in 96well plates, freeze replica plates and provide the clones for the isolation of DNA. The investigator is responsible for the genotyping identifying clones that underwent homologous recombination. Correctly targeted clones will be expanded, frozen in cryovials for the storage in LN2 and provided for the final genotype confirmation to ensure that proper homologous recombination occurred at both arms of the target vector. At this point we do not offer the preparation of the targeting construct, DNA for electroporation, isolation of DNA from resistant clones and molecular analysis of the clones.

Chromosomal abnormalities occurring in ES cells during gene targeting and prolonged culture can negatively affect the generation of germline chimeras. If there are more than 2 targeted clones identified, the simple chromosome counting may be helpful to select the best clones for aggregation or microinjection. We can prepare metaphase spreads and count the number of chromosomes per spread (minimum 20 spreads per clone). ES cell clones with at least 60% of the spreads containing 40 chromosomes would be selected for chimeras' production. We can also provide ES cell clones to Cytogenomics and Genome Resources Facility for the full karyotype analysis. Note that the euploidy of ES cell clone does NOT guarantee the germline but it is an effective method to exclude the clones that will not produce germline chimeras (Hughes ED, Qu YY, Genik SJ, Lyons RH, Pacheco CD, Lieberman AP, Samuelson LC, Nasonkin IO, Camper SA, Van Keuren ML, Saunders TL. 2007. Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line. Mamm Genome. 18:549-558)
We will prepare the targeted clones for aggregation or microinjection experiments co-ordinated within Transgenic Core. It is generally recommended to generate chimeras from two - three independent clones with the expectation that one or two will transmit through the germline.
See the Gene Targeting Service Outline for more details.

Guidelines:

  • Ideally gene targeting vectors should be based on genomic DNA that is isogenic to the mouse ES cell line that will be used in experiments to ensure high targeting frequency. R1 (129X1x129S1), G4 (129S6 x C57BL/6) and C2 (C57BL/6) ES cell lines are currently available from SLRI ES cell facility and W4 (129S6) obtained from Sick Kids Hospital ES cell facility.
  • After the isolation of genomic clones for the gene of interest and generation of a detailed restriction map, the targeting vector should be designed. Genomic regions that can serve as Southern blot probes to recognize targeting events should be tested on Southern blots of isogenic genomic DNA from ES cells. That will ensure future rapid genotyping of both ES cells and mice.
  • Gene targeting strategies including conditional 'floxed' alleles and generation of point mutations are described in multiple publications (see selected references below). For the generation of a simple null mutation, the following are brief guidelines:
    • The total amount of chromosomal homology is usually recommended to be between 4 and 8 kb with each arm being about half of the total amount of homology. A minimal targeting arm length on one side of 1 kb, with at least 2-3 kb on the other side or both of the targeting arms ~2 kb in size. There may be a significant advantage in using substantially longer targeting arms; however, large constructs are more difficult to manipulate.
    • A neomycin phosphotransferase (neo) gene expression cassette with the mouse. phosphoglycerate kinase (PGK) promoter and polyadenylation signal is used as a positive selectable marker for selection with G418. The neo cassette could be flanked by loxP or FRT sequences for its subsequent removal by Cre or FLPe recombinases (there is a concern regarding the repressor effect of the selectable marker on the genes in the vicinity of its insertion).
    • A herpes simplex virus thymidine kinase (TK) gene expression cassette can be used for negative selection with gancyclovir or FIAU. Single-selection vectors with PGK-Neo may have lower rate of homologous recombination in exchange for a better rate of germline transmission. The diphtheria toxin A-fragment gene can be efficiently used to negatively select non-homologous recombinants.
    • At least one unique restriction enzyme site must be present as a site for linearization of the final targeting vector. This site must be outside the homologous sequences, typically at the junction of one of the homologous arms and the plasmid backbone.
    • Restriction enzyme sites that can be used for the diagnosis of targeted events should be considered during the construction of the vector. A unique restriction enzyme site should be introduced by the positive selection cassette to use in a diagnostic digest with a probe external to the regions of homology.
    • Restriction enzymes that work efficiently on DNA samples prepared using 96-well plates must be used for the screening strategy. Correct gene targeting events must be identified using both 5' and 3' external probes.
    • PCR analysis may be suitable for analysis if appropriate control experiments have been done.
  • It is imperative that the methodology for detecting recombinant alleles has been worked out prior the initiation of experiments. Wild type ES cells in 96-well format can be provided as necessary.
  • Please be sure to develop a good genomic map of the targeted locus and unique probes that are external to the targeting construct BEFORE beginning gene targeting experiments. After extraction of the DNA from drug-resistant ES clones, there is only enough DNA for a single restriction digest and Southern analysis.
  • Note that the plates of frozen replica plates with ES cells can not be stored for more than 3-4 months. Delaying identification of targeted clones beyond that time risks the loss of the clones.

References

  1. Teratocarcinomas and Embryonic Stem Cells; A Practical Approach. Robertson EJ, ed., IRL Press at Oxford University Press, 1987
  2. Methods in Enzymology. Guide to Techniques in Mouse Development. 1993. Edited by Paul M. Wassarman and Melvin L. DePamphilis. Vold. 225.
  3. Manipulating the Mouse Embryo: A Laboratory Manual. Hogan B, Beddington R, Constantini F, Lacy E. 1994. Cold Spring Harbor Press. New York.
  4. Gene Targeting: A Practical Approach, 2nd Edition. Joyner AL, ed. 2000. IRL Press at Oxford University Press. New York.
  5. Manipulating the Mouse Embryo: A Laboratory Manual. Nagy, A, Gertsenstein, M, Vintersten, K, Behringer, R. 2003. Cold Spring Harbor Press. New York.

Service Charges

Service Mount Sinai and Sick Kids Hospitals and University of Toronto Faculty of Medicine
 External Canadian Academic Other External Academic For Profit Organizations
Gene targeting (per experiment) $5000 $6500 $8000 $10,000
Expansion of positive clones in addition to five included (per clone) $100 $130 $160 $200
Expansion of 96 well plate in 24 wells if required (per 24 well plate) $200 $260 $320 $400
Counting chromosomes (up to 3 clones) $300 $390 $480 $600
Counting chromosomes (each additional clone) $150 $195 $240 $300
Preparation of ES Cells for outsourced karyotype analysis (per clone) $100 $130 $160 $200
    The fees include:
  • Gene Targeting: up to three electroporations, selection, colony picking (up to 5 x 96 well plates), cryopreservation, replica plates, expansion of targeted clones, cryopreservation in vials, storage in LN2, cell pellets for the confirmation of the genotype (up to five positive clones, additional charge for each extra clone) and preparation of ES cells for ONE aggregation or microinjection.
  • Thawing and culture of ES cell clones, colcemid treatment, preparation of metaphase spreads and chromosome counting or cells delivery to Cytogenomics facility
    The fees do not include:
  • Karyotype analysis by The Cytogenomics and Genome Resources Facility. It can be charged directly or invoiced by TCP at cost.

How to Order

  1. Contact us and provide the gene-targeting strategy with the evidence of validated screening protocol tested on wild-type genomic DNA from ES cells, following the protocol for Preparation of DNA from 96well plates (PDF)
  2. Register for TCP LIMS account
  3. Once the strategy and the screening are approved, prepare DNA following the protocol for Preparation of DNA for electroporation of ES cells (PDF)
  4. Submit the service request: Login to LIMS and from the top menu go to Service Requests -> Request a TCP Service page, search with Department set to "TCP Transgenic Core and Specialty Resources", then select the service request
  5. Bring DNA to TCP with a gel photo of linearized vector attached to the printed service request form

Expansion of ES Cells Clones and Their Preparation for the Generation of Chimeras

Service Details      Service Charges      How To Order


Service Details

The evidence of murine pathogens screening is part of TCP requirements to test all biological materials prior to their introduction into SPF animals. PCR-based Alternative to MAP (mouse antibody production) testing offered at Research Animal Diagnostic Laboratory is used for screening ES cells clones used for generation of chimeras.

TCP Transgenic Core accepts ES cell clones from the following facilities:

  1. Approved Toronto ES cell facilities at Sick Kids Hospital; Samuel Lunenfeld Research Institute at Mount Sinai Hospital; CMHD Gene Trap and CMMR; TCP Transgenic Core.

    The biosafety cabinets (BSC) and incubators in these contained facilities are used exclusively for pathogen-tested ES cells and quality-controlled stocks and reagents. The cells leave the facility only as frozen vials or in the sealed tubes with aseptically prepared cell suspension. These facilities provide TCP Transgenic Core with the documentation showing that all new stocks of Primary Embryonic Mouse fibroblasts (MEF) and parental ES cell lines used for genetic manipulations are tested for mouse pathogens by RADIL Impact Profile I and quarterly with special panel designed by RADIL for TCP ('TCP IV'): RADIL Impact Profile IV + MNV and Ectro. Quarterly testing may include parental ES cell lines and/or selected genetically modified ES cell clone(s).

    ES cells from these facilities can be prepared for chimeras' experiments by:

  2. International Gene Trap and
    Knockout consortia: KOMP (at http://www.knockoutmouse.org or http://www.komp.org), EuCOMM, NorCOMM, TIGM

    All imported cells need to be tested by RADIL Impact Profile 'TCP IV' prior to the initiation of chimeras' experiments if the documentation of required pathogen testing is not provided. We can receive and expand untested ES cell clones in the quarantine BSC and send the sample (s) for pathogen-test (up to 5 clones from the same experiment can be pooled together for this test).

    Alternatively ES cells can be expanded by the investigator provided tested ES cell qualified reagents are used and TCP TG Core SOPs for the pathogen-test samples and preparation of ES cells for aggregation or microinjection are followed (See protocols for Preparation of ES cells for Aggregation and Preparation of ES Cells for Microinjection)

    NorCOMM ES cells ordered from CMMR: (http://www.cmmr.ca/reference/faqs_norcomm.html)

    CMMR can prepare ES cell clones for chimeras' experiment during their expansion and co-ordinate it with TCP Transgenic Core once the appropriate service requests are submitted.

    For more information on NorCOMM ES cell clones withdrawal as well as other CMMR services see http://www.phenogenomics.ca/services/cmmr/escell_services.html


Service Charges

Service Mount Sinai and Sick Kids Hospitals and University of Toronto Faculty of Medicine
 External Canadian Academic Other External Academic For Profit Organizations
Expansion of ES cell clones from other facilities (per clone) $300 $390 $480 $600
Preparation of ES cells for aggregation or microinjection
(per clone)		 $77 $100 $123 $154
    The fees include:
  • Clone expansion: receipt of frozen vials, expansion in tested culture conditions, cryopreservation (up to 5 vials per clone), LN2 storage, preparation of the sample for pathogen testing at RADIL and cell pellets for molecular analysis, if required as well as preparation of cells for ONE chimera experiment.
  • Preparation of ES cells for aggregation or microinjection: receipt of frozen vials with pathogen-tested ES cells, co-ordination of the experiment with Transgenic Core, thawing and culture in tested culture conditions, cryopreservation if required (up to 2 vials)
    The fees do not include:
  • Pathogen testing and the sample shipment to RADIL. It can be charged by RADIL directly or invoiced by TCP at cost.
  • Special Media if required for the clone expansion (e.g. RESGRO? ) - charged at cost

How to Order

  1. Contact us and provide the details of the clone(s), arrange for the direct shipment of frozen vials or bring the vials to TCP.
  2. Register for TCP LIMS account
  3. Submit the service request for Clone Expansion or Preparation of ES cells for experiment: Login to LIMS and from the top menu go to Service Requests -> Request a TCP Service page, search with Department set to "TCP Transgenic Core and Specialty Resources", then select necessary service request

Derivation of Novel ES Cell Lines

Service Details      Service Charges      How To Order


Service Details

ES cells derived from genetically modified mice represent important tools for analysis of the mutations especially when the phenotype is embryonic lethal at early stages of development. We have the expertise and the skills in handling mouse embryos and derivation of multiple ES cell lines established in Dr. A. Nagy laboratory (e.g. George S.H.L., Gertsenstein M., Vintersten K., Korets-Smith E., Murphy J., Stevens M.E., Haigh J.J., and Nagy A. Developmental and adult phenotyping directly from mutant embryonic stem cells. PNAS 2007, Mar 13, 104 (11): 4455-60).

We will plate 30-50 embryos, disaggregate the outgrowths, expand the colonies with ES cell morphology, freeze them in vials and provide the cell pellets for genotyping. We can use conventional methodology as well as the protocols based on more recent work:

  1. Schoonjans L, Kreemers V, Danloy S et al. Improved generation of germline competent embryonic stem cell lines from inbred mouse strains. Stem Cells 2003; 21:90 -97.
  2. Cheng J, Dutra A, Takesono A et al. Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis 2004; 39:100 -104.
  3. Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A. The ground state of embryonic stem cell self-renewal. Nature. 2008 May 22; 453(7194):519-23.
  4. Batlle-Morera L, Smith A, Nichols J. Parameters influencing derivation of embryonic stem cells from murine embryos. Genesis. 2008 Dec; 46(12):758-67.

Service Charges

Service Mount Sinai and Sick Kids Hospitals and University of Toronto Faculty of Medicine
 External Canadian Academic Other External Academic For Profit Organizations
Derivation of novel ES cell lines
(base fee per attempt)		 $300 $390 $480 $600
Expansion of derived ES cell lines
(per each new line)		 $77 $100 $123 $154
    The fees include:
  • Plating of blastocysts, disaggregation of the outgrowths, expansion of all clones with ES cell morphology, cryopreservation (up to 5 vials - charged per line), preparation of the sample for the pathogen testing if required
    The fees do not include:
  • The cost of superovulation and embryos collection (charged separately depending on the number of animals according to Transgenic Core fee schedule) - link to misc mouse service description
    Special Media if required (e.g. RESGRO? ) - charged at cost
    Pathogen testing and the sample shipment to RADIL. It can be charged by RADIL directly or invoiced by TCP at cost.s

How to Order

  1. 1.Contact us and provide the details on the strain, background, number of animals available etc
  2. Register for TCP LIMS account
  3. Submit the service request for Derivation of novel ES cell lines: Login to LIMS and from the top menu go to Service Requests -> Request a TCP Service page, search with Department set to "TCP Transgenic Core and Specialty Resources", then select necessary service request

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