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ES Cell Services



Gene Targeting

Service Details      Service Charges      How To Order


Service Details

The investigator is responsible for screening ES cell clones that underwent homologous recombination and has to have the genotyping strategy tested on wild type ES cells in 96 well format prior to the the experiments. . The facility 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. Correctly targeted clones will be expanded, frozen in cryovials for the storage in LN2 with cell pellets 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 drug-resistant clones and molecular analysis of the clones.

See the Gene Targeting Service Outline for more details and timeline. Once positive ES cell clones are confirmed , we will prepare them for aggregation or microinjection.

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/6NTac), C2 (C57BL/6NTac) and W4 (129S6) are currently available.
  • 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 cannot be stored longer 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.
  6. Gene Knockout Protocols: Second Edition, vol.530 Methods in Molecular Biology. R.Kuhn, W.Wurst (eds) Humana Press 2009
  7. Advanced Protocols for Animal Transgenesis: An ISTT Manual. S.Pease, T.L.Saunders (eds) Springer 2011

Service Charges

Service Mount Sinai &
Sick Kids
Hospitals
External Canadian Academic Other External Academic
Wild type ES cells in 96-well format provided to test genotyping strategy $53 $69 $85
Electroporation and isolation of antibiotic-resistant ES cell clones (per attempt - 2 cuvettes/maximum 2x 96 well plates) $1,591 $2,068
$2,545
Expansion of positive clones and new ES cell lines (per clone) - 2 vials and cell pellet $75 $97.50
$120
Additional vials or cell pellets (per vial/pellet) from cells in culture $37 $48
$59

How to Order

  1. PI and the lab contact register at TCP LIMS
  2. Contact us to submit 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) . We can provide 96-well plate with wild type ES cells.
  3. Once the strategy and the screening are validated, prepare DNA following the protocol for Preparation of DNA for electroporation of ES cells (PDF) and provide the Nanodrop profile
  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 for Gene Targeting
  5. Bring DNA prep to TCP with a gel photo of linearized vector


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

Service Details      Service Charges      How To Order


Service Details

ES cell clones ordered from the repositories of International Knockout Mouse Consortium (IKMC) (www.knockoutmouse.org/ ) can be shipped directly to TCP Transgenic Core.

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 IDEXX and Charles River Laboratories are 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; CMMR; TCP Transgenic Core and repositories of International Knockout Mouse Consortium (IKMC) - 1. Cell samples from these facilities are regularly tested and results are provided to TG core.
  2. 2. All ES cells originating from or propagated in other laboratories must be tested prior to the initiation of chimeras' generation experiments. TCP Transgenic Core offers a service to grow untested ES cell clones in a quarantine hood and incubator and send the samples for a test before generation of chimeras on a cost-recovery basis

Service Charges

Service Mount Sinai &
Sick Kids
Hospitals
External Canadian Academic Other External Academic
Expansion of ES cell clone from other facility (per clone) 15% discount for each additional clone from the same source expanded at the same time $318 $413 $509
ESC preparation for aggregation/microinjection (per clone)-includes re-freezing $106 $137 $169
Additional vials or cell pellets (per vial/pellet) from the cells in culture $37 $48 $59
2i ES cell culture medium (per 50 ml) for C57BL/6 ES cells $52 $52 $52
    The fees include:
  • ES cell clone(s) expansion: receipt of frozen vials, expansion in tested culture conditions, their cryopreservation , LN2 storage, preparation of the sample for pathogen testing if required, preparation of cell pellets for molecular analysis.
  • Preparation of ES cells for aggregation or microinjection: receipt of frozen vials with pathogen-tested ES cells, co-ordination of chimeras experiment, thawing and culture in tested culture conditions, cryopreservation

How to Order

  1. PI and the lab contact register at TCP LIMS
  2. Arrange for the direct shipment of frozen vials to TCP TG core or bring the vials to TCP.
  3. 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 necessary service request
    • TG core Expansion of ES cells
    • Preparation of ES cells for aggregation or microinjection


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. Andras Nagy's laboratory:
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
Gertsenstein M, Nutter LM, Reid T, Pereira M, Stanford WL, Rossant J, Nagy A. Efficient generation of germ line transmitting chimeras from C57BL/6N ES cells by aggregation with outbred host embryos. PLoS One. 2010 Jun 22; 5(6):e11260

We can use embryos from animals housed in TCP or other local facilities. We will plate the embryos, disaggregate the outgrowths, expand the colonies with ES cell morphology, freeze cells in vials or 96well replica plates depending on the total numbers and provide the cells for genotyping. Once the genotype is confirmed we will expand cell lines with required genotype. Recently we have been using culture conditions with 2i inhibitors as described:

  1. 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.
  2. Batlle-Morera L, Smith A, Nichols J. Parameters influencing derivation of embryonic stem cells from murine embryos. Genesis. 2008 Dec; 46(12):758-67.
  3. Nichols J, Jones K, Phillips JM, Newland SA, Roode M, et al. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat Med. 2009; 15:814-818.
  4. Sato H, Amagai K, Shimizukawa R, Tamai Y. Stable generation of serum- and feeder-free embryonic stem cell-derived mice with full germline-competency by using a GSK3 specific inhibitor. Genesis. 2009;47:414-422.

Service Charges

Service Mount Sinai &
Sick Kids
Hospitals
External Canadian Academic Other External Academic
Derivation of novel ES cell lines (base fee per attempt)- does not include expansion and cryopreservation $530 $689 $848
Expansion of newly derived ES cell lines (per line) - 2 vials and cell pellet $75 $97 $120
Additional vials or cell pellets (per vial/pellet) from cells in culture $36 $47 $58
2i culture medium (per 50 ml) for C57BL/6 ES cells $50 $50 $50
    The fees include:
  • Plating the embryos , disaggregation of the outgrowths,
  • Expansion of clones with ES cell morphology, their initial cryopreservation
  • Expansion of clones after genotype confirmation and their cryopreservation in vials.

How to Order

  1. PI and the lab contact register for TCP LIMS
  2. Contact us and provide the details on the strain, background, number and location of the animals in TCP.
  3. 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 for Derivation of novel ES cells.

 

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