Abstract
As little as a decade ago, generation of a single knockout mouse line was an expensive and time-consuming undertaking available to relatively few researchers. The International Knockout Mouse Consortium, established in 2007, has revolutionized the use of such models by creating an open-access repository of embryonic stem (ES) cells that, through sequential breeding with first FLP1 recombinase and then Cre recombinase transgenic mice, facilitates germline global or conditional deletion of almost every gene in the mouse genome. In this Case Study, we describe our experience using the repository to create mouse lines for a variety of experimental purposes. Specifically, we discuss the process of obtaining germline transmission of two European Conditional Mouse Mutagenesis Program (EUCOMM) “knockout-first” gene targeted constructs and the advantages and pitfalls of using this system. We then outline our breeding strategy and the outcomes of our efforts to generate global and conditional knockouts and reporter mice for the genes of interest. Line maintenance, removal of recombinase transgenes, and cryopreservation are also considered. Our approach led to the generation of heterozygous knockout mice within 6 months of commencing breeding to the founder mice. By describing our experiences with the EUCOMM ES cells and subsequent breeding steps, we hope to assist other researchers with the application of this valuable approach to generating versatile knockout mouse lines.
Highlights
The humble laboratory mouse has had a home in scientific research since the 15th century, and continues to prove an indispensable resource for investigators wishing to unravel the biology of mammalian development, genetics, physiology, and pathology
Researchers who are eager to commence animal studies would benefit from a library of pre-mutated mouse embryonic stem (ES) cells; a need that was addressed last decade with the establishment of the International Knockout Mouse Consortium (IKMC)
Development of the various targeting vectors is described in detail in the original paper [5]; here, we provide an outline of how this approach would be applied to a prototypical GOI containing three exons with exon 2 having been identified as “critical” for gene function
Summary
The humble laboratory mouse has had a home in scientific research since the 15th century, and continues to prove an indispensable resource for investigators wishing to unravel the biology of mammalian development, genetics, physiology, and pathology. This is largely due to the mouse’s small size and resource demand, short generation time, and suitability for breeding within a laboratory environment [1]. Prioritization of targets has been based upon community demand, expert committees, and the perceived importance of individual genes, with little overlap between programs [3] In our case, both of our genes of interest were targeted and available through EUCOMM, so we will focus this review on the gene targeting approach specific to EUCOMM ES cells. As of 2012, more than 17,400 ES cell lines had been generated by the IKMC for targeted gene deletion [4]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
