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pronuclear-injection.jpgThe classical technology used for generating transgenic mouse lines is based on the random integration of exogenous DNA into the genome which occurs when DNA is injected into the pronucleus of a fertilized oocyte. Transgenic mice which have integrated the transgenic construct are known as "founder mice" and each founder mouse can be bred with wild-type mice to establish different "lines" of transgenic mice. As the genomic integration event is random, expression characteristics vary between transgenic lines, and subsequently, each transgenic line is bred and analysed independently. Depending on the aim of the experiment, these inter-line variations can be useful as the different lines may express the transgene at different levels, thus allowing a correlation between expression level and phenotype.

The transgenic core provides a high quality injection service for transgenic constructs in a variety of genetic backgrounds and is able to guarantee the generation of transgenic founders. The service includes the preparation and purification of the transgene and the screening of putative founder pups by PCR analysis.

Pronuclear microinjection is a method which is based on the integration of the transgenic construct at random into the genome. This random insertion is associated with a lack or predictability which can complicate the comparison of different transgenic lines and even different individual organisms.

  • The random insertion of the DNA construct can result in the mutagenesis of endogenous genes
  • Transgene expression is very dependent upon the integration site. So called "position effects" can lead to silencing or inconsistent or unexpected ("ectopic") expression patterns
  • The DNA construct integrates often in multi-copy tandem arrays which are associated with non-homogenous expression ("variegation"). Within a particular target tissue, not every cell expresses the transgene which can complicate phenotypic analysis
  • As the locus of integration is initially uncharacterized, heterozygous and homozygous trans­genic mice cannot be easily distinguished

Due to these unpredictable factors, the analysis of several independent lines is necessary to conclude that the observed phenotype is associated with the transgene and not due to non-specific effects. When planning such an experiment, it is important to consider that the housing, breeding, characterisation and phenotyping of the independent lines increases the downstream project costs considerably.

Service

  • Removal of vector sequences and the purification of the transgenic construct
  • Pronuclear microinjection
  • Transfer of microinjected embryos and housing of the resulting pups until 6 week
  • Establishment of a robust PCR based screening strategy
  • Identification of transgenic founder animals by PCR
  • Full documentation and delivery of at least three independent founder animals