Generation of Chimeras

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ES cells

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

 

Aggregation and Microinjection

Based on 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 R1 (129X1x129S1) and G4 (129S6 x C57BL/6), W4 (129S6) ES cell clones.. This method also works with C57BL/6 ES cell lines grown in specific culture conditions.
http://www.ncbi.nlm.nih.gov/pubmed/20582321

We also offer C57BL/6 morula/blastocysts microinjections as a classical method to generate chimeras from 129-derived ES cells e.g. E14 (129P2) where it is beneficial for better visualization of coat colour chimerism.

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. Male chimeras with moderate and high ES cell contribution are tested for germline transmission by the investigator and the results provided to Transgenic Core.

Alternatively Transgenic Core can perform test- -breeding for germ line transmission. 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 (e.g. TS or XEN 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.

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 we can use EGFP as 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.

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 mouse strain is available as donors of diploid and tetraploid host embryos for various chimeric combinations. When mutant diploid or tetraploid embryos are requeired, 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 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)	$2035	$2646	$3256
Aggregation with tetraploid ICR or EGFP embryos (per attempt)	$2035	$2646	$3256
C57BL/6 blastocyst/morula microinjection (per attempt)	$2255	$2932	$3608
ICR blastocyst/morula microinjection (per attempt)	$2195	$2854	$3512
Breeding chimeras for germline (per chimera) - does not include cage per diem, animal purchase and tissue biopsies	$50	$60	$85
The fees include: purchase of embryo donor animals and their housing prior to the experiment one aggregation or microinjection experiment (up to two clones) transfer of microinjected or aggregated embryos into pseudopregnant recipients test-breeding for germline (if requested) The fees do not include: animal housing (per diem) charges for recipients and their offspring from the time of embryo transfer surgery health monitoring (foster mothers) export fees and shipping charges animal cost, cage per diem and tissue biopsies for germline test-breeding (if requested)

How to Order

1. Refer to Policies and Procedures for the required documentation prior to the initiation of the experiment and Service Agreements
2. Follow the guidelines above about pathogen-testing of ES cells
3. Register for TCP LIMS account
4. 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