Generation of Chimeras
Service Details
Service Charges
How To Order
Service Details
ES cells
Pathogen Test
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 cell clones used for generation of chimeras. TCP Transgenic Core accepts ES cell clones from the following facilities:
- 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 investigator (using the protocols: Preparation of ES Cells for Aggregation (PDF), Preparation of ES Cells for Blastocyst Microinjection (PDF)) OR provided to Transgenic Core in frozen vials by submitting Preparation of ES cells service request
- International Gene Trap and Knockout consortia: KOMP (at http://www.knockoutmouse.org or http://www.komp.org), EuCOMM, NorCOMM, and 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 (using the protocols: Preparation of ES Cells for Aggregation (PDF), Preparation of ES Cells for Blastocyst Microinjection (PDF))
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
Karyotype
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. Transgenic Core 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)
It is generally recommended to generate chimeras from two - three independent ES cell clones with the expectation that one or two will transmit through the germline).
Aggregation and Microinjection
Based on more than 16 years experience at Samuel Lunenfeld Research Institute Mount Sinai Hospital Transgenic Facility, TCP Transgenic Core continues to offer outbred morula aggregations as a primary method to generate chimeras with R1 (129X1x129S1) and G4 (129S6 x C57BL/6) ES cell clones. This method is easier than microinjection and requires cheaper outbred ICR embryo donors. Although we have more limited experience with these cells this method also works with W4 (129S6), E14 (129P2) and some C57BL/6 ES cell lines grown is specific culture conditions.
We also offer C57BL/6 morula/blastocysts microinjections as a classical method to generate chimeras from 129-derived ES cells
Aggregated or microinjected embryos are transferred into pseudopregnant recipients and allowed to reach term. Litters are monitored and evaluated for coat color chimerism. Chimeras are transferred to the investigator after weaning and completion of the health monitoring. Male chimeras with moderate and high ES cell contribution are tested for germline transmission by the investigator and results provided to Transgenic Core.
Alternatively Transgenic Core offers breedng for germ line transmission service. Animals for the test-breeding purchased from in house breeding colony as well as per diem cage charges are billed directly to the investigator in addition to the fees listed below.
Other types of stem cells can be injected into ICR morula or blastocysts for the assessment of their developmental potential. The evidence of murine pathogens screening should be provided to TCP as described above for ES cells. Please, contact us for more details.
Tetraploid Complementation Assay
The contribution of tetraploid embryos is primarily restricted to extraembryonic tissues. Complementation of tetraploid embryos with ES cells capable of contributing to embryonic lineages results in the production of embryos, nearly 100% derived from ES cells. This approach can be used to study and bypass extraembryonic defects of mutations, and to produce mutant embryos directly from ES cells without breeding for the analysis of their phenotype. When F1-hybrid ES cells are used for aggregation with tetraploid embryos, it is possible to generate fully ES-cell derived animals carrying mutations directly from ES cells and speed up traditional breeding by one generation.
Tetraploid embryos are produced using an electric pulse to fuse two blastomeres of 2-cell stage embryos and incubated overnight. The following day, two tetraploid embryos at 4-cell stage are aggregated with ES cells. Aggregates are cultured overnight and transferred into pseudopregnant females that can be dissected at desired embryonic stage or left to term. To confirm ES cell origin of embryos dissected at mid-gestation we use EGFP, EYFP, or RFP as tetraploid host embryos for aggregation with genetically modified ES cell clones. Wild type ICR tetraploid embryos are used for aggregations with ES cell clones that are left to term.
Chimeric Analysis
Aggregation technique provides a useful approach for the analysis of mutant phenotypes using chimeras. Lack of, or biased contribution of mutant cells to certain lineages and tissues in chimeras between mutant and wild type diploid embryos may answer questions about cell autonomy of the mutant phenotype. Wild-type tetraploid embryos can contribute to the defective trophoblast and primitive endoderm derivatives of mutant embryos with extraembryonic phenotype and provide access to the embryonic phenotype.
Mutant ES cells can be aggregated with fluorescent diploid embryos and wild type ES cells with mutant diploid or tetraploid embryos for transient analysis at mid-gestation. EGFP, EYFP, and RFP mouse strains are available as donors of diploid and tetraploid host embryos for various chimeric combinations. For the cases that require mutant diploid or tetraploid embryos, the investigator will need to provide sufficient number of males and females at the proper age. Please, contact us for more details.
Additional information on the aggregation method, tetraploid complementation assay, chimeric analysis and their applications can be found in these publications.
References
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 |
| Aggregation with tetraploid OR diploid ICR, EGFP, EYFP, or RFP embryos (per attempt) |
$2035 |
$2646 |
$3256 |
$4070 |
| C57BL/6 blastocyst/morula microinjection (per attempt) |
$2255 |
$2932 |
$3608 |
$4510 |
| ICR blastocyst/morula microinjection (per attempt)) |
$2195 |
$2854 |
$3512 |
$4390 |
| Breeding chimeras for germline (per ES cell line) - NOT including animal purchase, per diem and tissue biopsies |
$200 |
$260 |
$320 |
$400 |
| Mouse identification and tissue biopsies (per cage) |
$10 |
$13 |
$16 |
$20 |
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How to Order
- Refer to Policies and Procedures for the required documentation prior to the initiation of the experiment and Service Agreements
- Follow the guidelines above about pathogen-testing of ES cells
- Register for TCP LIMS account
- 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 appropriate service request
- 1.If you require Clone Expansion or Preparation of ES cells for experiment from the frozen vial, submit the service request accordingly and provide frozen vial(s) to the Transgenic Core.
- Alternatively, prepare the cell suspension according to the protocols and bring it to TCP on the day of the experiment at the time requested by the Transgenic Core Staff: