Generation of Chimeras

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Service Details

ES cells

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

 

Aggregation and Microinjection

Based on the experience at Samuel Lunenfeld Research Institute Mount Sinai Hospital Transgenic Facility since 1990s, TCP Transgenic Core continues to offer outbred albino morula aggregations as a primary method to generate chimeras with ES cell clones from following parental ES cell lines: R1 (129X1x129S1), G4 (129S6 x C57BL/6NTac), W4 (129S6) and C2 (B6NTac) genetically modified at the local ES cell facilities. We use aggregation method with C57BL/6 ES cell lines from International Knockout Mouse Consortium (IKMC) in combination with specific culture conditions with inhibitors, for more details see http://www.ncbi.nlm.nih.gov/pubmed/20582321

Since 2011 TCP Transgenic Core participates in NorCOMM2 and KOMP2 - DTCC helping to generate mouse models from IKMC resources for phenotyping - the effort co-ordinated by the International Phenotyping Consortium (IMPC).

Also see http://www.phenogenomics.ca/transgenics/index.html

We also offer C57BL/6 microinjections as a classical method to generate chimeras from 129-derived ES cells e.g. E14 (129P2).

Aggregated or microinjected embryos are transferred into pseudopregnant recipients and allowed to reach term. Litters are monitored and evaluated for coat colour chimerism. Chimeras are transferred to the investigator after weaning and completion of the health monitoring or can be tested for germline transmission as a service. Animals for the test-breeding purchased from TCP breeding colony or commercial as well as per diem cage charges during test-breeding are billed directly to the investigator in addition to the fees listed below.

Animals for the test-breeding purchased from in house breeding colony or commercial 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 (e.g. TS, XEN, iPS cells) can be aggregated or microinjected for the assessment of their developmental potential. The evidence of murine pathogens screening should be provided to TCP as described for ES cells

Tetraploid Complementation Assay and Chimeric Analysis

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. It is possible to generate fully ES-cell derived animals carrying mutations directly from tested F1-hybrid 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 EGFP transgenic mice can be used as embryo donors for tetraploid host embryos for aggregation with genetically modified ES cell clones. Wild type ICR tetraploid embryos are typically used for aggregations with F1-hybrid ES cell clones that are left to term.

Chimeras provide a useful approach for the analysis of mutant phenotypes. 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 EGFP diploid embryos and wild type ES cells with mutant diploid or tetraploid embryos for transient analysis at mid-gestation. EGFP mouse strain is available as donors of diploid and tetraploid host embryos for various chimeric combinations. When mutant diploid or tetraploid embryos are required for the project, the investigator will provide sufficient number of males and females at the proper age. Please, contact us for more details to co-ordinate such experiments.

Additional information on the aggregation method, tetraploid complementation assay, chimeric analysis and their applications can be found in the publications below.

References

1. Tarkowski, A. K., Witkowska, A., and Opas, J. Development of cytochalasin in B-induced tetraploid and diploid/tetraploid mosaic mouse embryos. J Embryol Exp Morphol, 1977 4147-64.
2. Kubiak, J. Z. and Tarkowski, A. K. Electrofusion of mouse blastomeres. Exp Cell Res, 1985 157(2) 561-6.
3. Nagy, A., E. Gocza, E. Merentes-Diaz, V.R. Prideaux, E. Ivanyi, M. Markkula and J. Rossant. Embryonic stem cells alone are able to support fetal development in the mouse. Development 1990 110, 815-821.
4. Nagy, A., J. Rossant, R. Nagy, W. Abramow-Newerly and J.C. Roder Derivation of completely cell culture-derived mice from early passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 1993 90, 8424-8428.
5. Wood, S.A., N.D. Allen, J. Rossant, A. Auerbach and A. Nagy. Non-injection methods for the production of embryonic stem cell-embryo chimeras. Nature, 1993, 365, 87-89.
6. Nagy A, Rossant J. Targeted mutagenesis: analysis of phenotype without germ line transmission. J Clin Invest. 1996 Mar 15; 97(6):1360-5. Review.
7. Nagy A. and Rossant J. Chimaeras and mosaics for dissecting complex mutant phenotypes. Int J Dev Biol. 2001 45: 577-582
8. Tam PP, Rossant J. Mouse embryonic chimeras: tools for studying mammalian development. Development. 2003 Dec; 130(25):6155-63. Review.
9. Kunath T, Gish G, Lickert H, Jones N, Pawson T, Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol. 2003 May; 21(5):559-61.
10. Vintersten K, Monetti C, Gertsenstein M, Zhang P, Laszlo L, Biechele S, Nagy A. Mouse in red: Red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis. 2004 Dec 9;40(4):241-246
11. Lickert H, Cox B, Wehrle C, Taketo MM, Kemler R, Rossant J.. Dissecting Wnt/beta-catenin signaling during gastrulation using RNA interference in mouse embryos. Development. 2005 Jun; 132(11):2599-609
12. Takeuchi JK, Mileikovskaia M, Koshiba-Takeuchi K, Heidt AB, Mori AD, Arruda EP, Gertsenstein M, Georges R, Davidson L, Mo R, Hui CC, Henkelman RM, Nemer M, Black BL, Nagy A, Bruneau BG. Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development. Development. 2005 May; 132(10):2463-74
13. Georgiades P, Rossant J. Ets2 is necessary in trophoblast for normal embryonic anteroposterior axis development. Development. 2006 Mar;133(6):1059-68. Epub 2006 Feb 15.
14. 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. 2007 PNAS 10.1073/pnas.0609277104
15. Takeuchi JK, Lickert H, Bisgrove BW, Sun X, Yamamoto M, Chawengsaksophak K, Hamada H, Yost HJ, Rossant J, Bruneau BG. Baf60c is a nuclear Notch signaling component required for the establishment of left-right asymmetry. Proc Natl Acad Sci U S A. 2007 Jan 16; 104(3):846-51.
16. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A. piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 2009 Apr 9; 458(7239):766-70.
17. Cox BJ, Vollmer M, Tamplin O, Lu M, Biechele S, Floss T, Gertsenstein M, van Campenhout C, Kühn R, Wurst W, Lickert H, Rossant J. Phenotypic annotation of the mouse X chromosome. Genome Res. 2010 Aug; 20(8):1154-64

Service Charges

Service	Mount Sinai & Sick Kids Hospitals	External Canadian Academic	Other External Academic
Aggregation with diploid ICR embryos (per attempt)	$2204	$2865	$3527
Aggregation with tetraploid ICR or EGFP embryos (per attempt)	$2369	$3080	$3790
C57BL/6 morula microinjection (per attempt)	$2204	$2865	$3527
ICR morula microinjection (per attempt)	$2204	$2865	$3527
Breeding chimeras for germline (per chimera) - does not include cage per diem, animal purchase and tissue biopsies	$52	$68	$83
The fees include: purchase of embryo donor animals and their housing prior to the experiment one aggregation or microinjection experiment (up to two clones) - please, see service agreement for details ChimServAgr_Apr2013.pdf transfer of microinjected or aggregated embryos into pseudopregnant recipients (up to 10 recipients) test-breeding for germline (optional, if requested) The fees do not include (extra charges apply): animal housing (cage per diem) charges for recipients and their offspring from the time of embryo transfer surgery health monitoring test of fosters, one per clone animal cost, cage per diem and tissue biopsies during germline test-breeding export fees and shipping charges

How to Order

1. Refer to Policies and Procedures for the required documentation prior to the initiation of the experiment and Service Agreements including guidelines on pathogen-testing of ES cells
2. PI and the lab contact register at TCP LIMS
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 microinjection or aggregation (diploid or tetraploid embryos)