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The Hospital for Sick Children and Mount Sinai Hospital share a legacy of pioneering work in mouse genetics, mutagenesis, and functional genomics. Seminal advances in transgenesis, gene trapping, homologous recombination, and genetic toolbox development by Janet Rossant, Alex Joyner, and Andras Nagy in the late 1980s established a foundation of excellence in mouse models. This enabled leadership and contributions to the first genomic approaches to mutagenesis like the global Gene Trap and Knockout Mouse Consortia from the late 1990's to mid 2000's.

The TCP Transgenic Core is an amalgamation of the Transgenic Facilities at Mount Sinai Hospital and The Hospital for Sick Children. These facilities have been operating since early 1990s and have pioneered innovative technologies such as the production of mouse ES cell chimeras using the aggregation method and Tetraploid Complementation Assay.1-3 See more recent examples of this technology's applications in following selected publications 4-11 . The TCP Transgenic core continues to maintain a close affiliation with the laboratories of Dr. Janet Rossant and Dr. Andras Nagy.

The Transgenic Core is integrated with the Molecular Biology Core into Mutant Model Production Core. It produces >250 model per year for individual users and international projects.

TCP Mutant Model Production Core provides a range of services generating Genetically Engineered Mouse Models (GEMM) for investigators from TCP member hospitals, the University of Toronto, external academic and for-profit institutions. We offer following services:

Molecular Biology Core »

  • Vector design and construction for the production of knock-out, conditional, or knock-in alleles or transgenic vectors
  • Genetic quality control: Screening ES cells for germline quality using chromosome-specific quantitation PCR probes. Screening ES cells and/or founder mice for the mutation of interest, genome-wide screening for off-target mutations after in vivo genome editing, development and validation of genotyping assays, assessment of targeting accuracy and allele sequencing, and validation of strain background.
  • Design and synthesis of reagents for genome editing using Cas9 RNA guided nucleases (Cas9-RGN) for generation of Cas9-RGN mediated models such as targeted deletions, non-homologous end joining (NHEJ) mediated insertion and deletion (indel) null alleles and point mutations

Contact Dr. Lauryl Nutter for more details:

Transgenic Core »

Genetically modified ES cells for generation of chimeras are provided by local Toronto ES cell facilities, The International Gene Trap Consortium including the Gene Trap Resource at The Centre for Modeling Human Disease (CMHD) and International Knockout Mouse Consortium (IKMC). CMHD gene trap and NorCOMM targeted clones are distributed by the Canadian Mouse Mutant Repository (CMMR) located at TCP.

We are integral parts of the Genome Canada funded NorCOMM2 and the NIH-funded KOMP2-DTCC projects since 2011. These projects are parts of the International Mouse Phenotyping Consortium (IMPC), a collaboration of 13 international production and phenotyping centres.

We have been successfully using outbred morula aggregation for many years and more recently reported combining this expertise with culture of C57BL/6N ESC in medium containing Knockout Serum Replacement (KOSR) and 2i inhibitors: MEK (PD0325901) and GSK3 (CHIR99021). See our publication 12 for more details on culture conditions: http://www.ncbi.nlm.nih.gov/pubmed/20582321

Germline transmission (GLT) rate from targeted C57BL/6 ES cells (July 2014 update) using outbred CD-1 (ICR) morulae

Number of ESC clones aggregated Number of ESC clones microinjected
Parental ES cell line Total With chimeras >50% Complete GLT (% per # complete) Total With chimeras >50% Complete GLT (% per # complete)
C2* 94 90 (96%) 92 71 (77%) 2 2(100%) 1 0
JM8 4 4 (100%) 4 3 (75%) 2 1(50%) 2 0
JM8.F6 14 13 (93%) 13 5 (38%) 13 10(77%) 13 4(31%)
JM8.N4 51 44 (86%) 50 31 (62%) 10 8 (80%) 10 2 (20%)
JM8A1.N3 25 23 (92%) 24 8 (33%) 34 31 (91%) 34 12 (35%)
JM8A3.N1 238 173 (73%) 231 77 (33%) 99 86 (87%) 94 37 (39%)
VGB6 38 33 (87%) 33 22 (67%) 12 12 (100%) 7 4 (57%)
JM8A3 2 1 (50%) 1 0
TIGM 4 3 (75%) 4 2 (50%)
Total 470 384 (82%) 452 219 (48%) 172 150 161 59 (37%)

* C57BL/6NTac-C2 is the ES cell line used by TCP Transgenic Core, the data from our gene targeting services using C2 are combined with NorCOMM and NorCOMM2 production pipelines.

 

C57BL/6N ES cell chimeras generated by aggregation with outbred ICR host embryos


Selected References

  1. 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. (http://dev.biologists.org/cgi/reprint/110/3/815)
  2. 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. (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=8378314)
  3. Wood, S.A., N.D. Allen, J. Rossant, A. Auerbach and A. Nagy. Non-injection methods for the production of embryonic stem cell-embryo chimaeras. Nature, 1993, 365, 87-89.
  4. 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.
  5. 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
  6. 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
  7. 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
  8. George S.H.L., Gertsenstein M., Vintersten K., Korets-Smith E., Murphy J., Stevens M.E., Haigh J.J., and Nagy A. (2007) Developmental and adult phenotyping directly from mutant embryonic stem cells. 2007 PNAS 10.1073/pnas.0609277104
  9. 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
  10. 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
  11. 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
  12. 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

For more information, please contact:

Marina Gertsenstein
The Transgenic Core
The Toronto Centre for Phenogenomics
25 Orde Street, Toronto, ON M5T 3H7
Tel: 647-837-5811 x4302
Fax: 647-837-5834
Email:

 

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