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Mouse model generation

The MLC offers a wide range of services for the production of genetically modified mice tailored to the needs of both internal and external investigators. We also provide advice about the transgenic strategies (pronuclear injection and gene targeting) and can facilitate the sourcing of mutant embryonic stem (ES) cell clones. Please see below for the full list of these services, and contact us to make an enquiry.

RGEN/CRISPR mediated mutagenesis

A genomic sequence is targeted by a Cas9 or Cas9 D10A/guide RNA complex. The resulting DNA cut is repaired by the cell machinery through NHEJ, giving rise to a deletion or, if a donor DNA was co-delivered, through homologous recombination. The resulting mice are mated and the targeted mutation is transmitted to offspring. This service includes:

  • Design and preparation of reagents (cas9/nickase/gRNAs/donors)
  • Production of embryos and pseudo-pregnant females
  • Micro-injection of embryos
  • Oviduct transfer of injected embryos to pseudo-pregnant foster mothers
  • Tissue biopsies of mice raised from injected embryos
  • Characterisation of endonuclease mediated mutations in F0
  • Breeding of F0 mice for the establishment and maintenance of genetically modified mouse lines, including tissue biopsies and identification of transgenic animals

Gene targeting

ES cells are electroporated with a targeting construct and colonies isolated. The clones are screened, amplified, quality controlled and converted to mice by microinjection into blastocysts. Chimera are obtained and mated for transmission of the genetic modification. This service includes:

  • Purification and preparation of targeting constructs
  • Targeting of ES cells or transient transfection of targeted ES clones for cre or flp driven recombination
  • Screen of ES cell colonie
  • Amplification of positive colonies and quality control by Southern blot and karyotyping
  • Production of blastocysts and pseudo-pregnant females
  • Injection of ES cells into blastocysts
  • Uterine transfer of injected blastocysts to pseudo-pregnant foster mothers
  • Breeding for the establishment and maintenance of genetically modified mouse strains including tissue biopsies and identification of transgenic animals
  • We routinely employ ES cells of 129 or C57BL/6N origin. Other backgrounds can be employed for specific projects

ES cell microinjection

Mutated ES cells can be obtained from a variety of sources. The cells are imported, amplified, quality controlled and converted into mice by injecting them into blastocysts. Chimera are obtained and mated for transmission of the genetic modification. Our services include:

  • Import of ES cells
  • Amplification of stock
  • Quality control of targeting event (Southern blot, PCR and sequencing)
  • PCR based MAP testing
  • Production of blastocysts and pseudo-pregnant females
  • Injection of ES cells into blastocysts
  • Uterine transfer of injected blastocysts to pseudo-pregnant foster mothers
  • Breeding for the establishment and maintenance of genetically modified mouse strains including tissue biopsies and identification and quality control of transgenic animals

Find mutated alleles by searching the IMPC web portal and the International Gene Trap Consortium.

Pronuclear injection

A DNA construct can be injected into 0.5-day-old fertilised embryos to produce transgenic mice containing the DNA in every cell. The DNA construct typically consists of a gene of interest that is expressed in a certain range of tissues. The resulting mice are mated and the DNA construct is then transmitted to offspring. Our services include:

  • Purification and preparation of construct for microinjection (e.g. plasmid, BAC, PAC)
  • Production of embryos and pseudo-pregnant females
  • Pronuclear Injection of embryos
  • Oviduct transfer of injected embryos to pseudo-pregnant foster mothers
  • Harvest and preparation of FO transgenic embryos
  • Tissue biopsies of mice raised from injected embryos
  • Breeding for the establishment and maintenance of genetically modified mouse strains including tissue biopsies for identification and characterisation of transgenic animals, including transgene copy counting
  • On request: Specific genetic background - please contact us to discuss the appropriate genetic background for your project