Abstract
With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.
Highlights
The International Mouse Phenotyping Consortium (IMPC) (Ayadi et al 2012; Brown and Moore 2012a, b; Laughlin et al 2012; Ramırez-Solis et al 2012; White et al 2013) is a large scale international consortium whose aim is to generate and primary phenotype a knockout mouse strain for each of the *20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium (IKMC) (Bradley et al 2012; Skarnes et al 2011)
Several large centralised repositories have been established around the world, including the Infrafrontier/European Mutant Mouse Archive (EMMA) (INFRAFRONTIER Consortium 2014; Wilkinson et al 2010), the KnockOut Mouse Project Repository (KOMP) (Lloyd 2011), the Jackson Laboratory Repository (Jax) (Ostermeier et al 2008), The Center for Animal Resources and Development (CARD) (Nakagata and Yamamura 2009) and the Riken Bio Resource Center (Yoshiki et al 2009), which provide cryopreserved material or live mice to receiving laboratories
We tested a much improved quality control method to determine the ability to recover a mutant mouse strain which has been stored in a cryopreserved form
Summary
The International Mouse Phenotyping Consortium (IMPC) (Ayadi et al 2012; Brown and Moore 2012a, b; Laughlin et al 2012; Ramırez-Solis et al 2012; White et al 2013) is a large scale international consortium whose aim is to generate and primary phenotype a knockout mouse strain for each of the *20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium (IKMC) (Bradley et al 2012; Skarnes et al 2011). A typical validation will involve thawing cryopreserved sperm to ensure fertilisation post-thaw can be achieved at a predetermined level ([10 % of treated eggs at EMMA) and can produce viable embryos This requires an in vitro fertilisation (IVF) procedure followed by surgical embryo transfer to pseudopregnant recipient females, pregnancy with births, and confirmation of the expected mutant genotypes from the resultant litter usually from tissue derived from an ear clip. We have successfully investigated a new simple and efficient methodology that involves the viability testing and genotyping directly on individual preimplantation blastocyst-stage embryos generated from fresh IVF or from frozen/thawed 2-cell stage embryos originally produced by IVF This new approach has major ethical advantages by reducing greatly the number of animals used in the QC process. It ensures that QC is processed faster, more robustly, and with significant reductions in cage space, technical resources, and overall cost
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