Overview

Mouse models of human cancer have had a profound impact on our current understanding of mechanisms of tumorigenesis and the pathways regulated by cancer-related genes. These models hold the promise of serving as critical tools in the discovery and testing of novel therapeutics to be used in the treatment and prevention of cancer.

The Transgenic Mouse Model Laboratory (TMML) provides assistance to the scientific community with the generation of genetically engineered mice (GEM), offering a complete array of technologies aimed at the successful generation of transgenic and gene-targeted mice, their expansion and cryopreservation.

Generation of transgenic mice by pronuclear microinjection:

  • Consultation with transgene design
  • Purification of transgene from vector sequences for microinjection
  • Microinjection of the transgene into the pronucleus of fertilized mouse eggs
  • Genotypic characterization of transgenic founder mice by Southern analysis
  • Guarantee of 3 independent founder mice for each transgene
  • Delivery of transgenic founders within 3 months of receipt of the transgenic vector
  • Establishment and cryopreservation of founder lines

The production of transgenic mice by pronuclear injection is an all-encompassing service, covering all the steps from receipt of a recombinant DNA construct containing the transgene of interest to the delivery of breeding-age transgenic founder mice. Production of GEMs is routinely performed in inbred backgrounds such as C57BL/6NCr and FVB/NCr, but on demand and for an additional charge, may also be produced in inbred backgrounds including Balb/C AnCr, NOD/SCID NCr, and SCID/NCr. TMML maintains a 100% success rate in the generation of transgenic mice, having introduced in excess of 1450 recombinant DNA constructs in the germline of mice. Vectors processed include plasmid DNA, cosmid, Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC).

Generation of Knock out and knock in by gene targeting:

  • Consultation with the targeting vector design
  • Gene targeting by electroporation into mouse embryonic stem cells (mESC)
  • Identification of homologous recombination events by Southern analysis
  • Karyotyping for selection of two clones for blastocyst microinjection to generate chimeras
  • Breeding of chimeras to ensure their capability for contribution to germline
  • Project completed within 6-10 months from initiation of gene targeting
  • Guarantee of germline transmission

The gene targeting service is an all-inclusive service that initiates with electroporation of the vector of interest into the embryonic stem cells. The laboratory commonly uses E14TG2A.4 mouse ESC, a stable cell line derived from 129P2/OlaHsd, with confirmed germline transmission capability. The C57BL/6NCrf ES cells, generated and established by the Transgenic Mouse Model (TMM) Laboratory, using C57BL/6NCr inbred strain, is also available for the gene targeting experiments. Following electroporation, homologous recombination events are identified through genotyping by Southern analysis. Karyotyping will be performed on successfully targeted clones to ensure genetic integrity. Two clones with normal karyotype will be selected for microinjection into wildtype blastocysts from the appropriate background, to generate high percentage chimeras, judged by percentage of coat color contribution from the ESC. Selected chimeric males will be placed in breeding to ensure germline transmission. Genotypic analysis on the first-generation progeny is performed by Southern blot analysis. The success of the comprehensive gene targeting projects, performed by TMML, is defined by confirmation of germline transmission.

Publications

For additional reference, please see a list of Publications relevant to research in the Transgenic Mouse Model Laboratory.