Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study

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.

Orphan receptor GPR37L1 contributes to the sexual dimorphism of central cardiovascular control

“…However, the mouse strain, sex, age, heart rate, and depth of anesthesia used for BP measurement were not indicated, and BP was not compared to appropriate wild-type controls. Here, we sought to definitively establish whether GPR37L1 regulates BP using GPR37L1 null mice that we generated and maintained on a pure C57BL/6J background [ 9 , 10 ]. We report that GPR37L1 is indeed involved in BP regulation but in a sexually dimorphic manner.…”

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells

“…The International Knockout Mouse Consortium (IKMC), and participating organizations, are using both targeted deletion and knockout-first allele strategies to generate embryonic stem (ES) cell lines with both knockout and conditional knockout potential ( Friedel et al 2007 ; Lloyd 2011 ). To date, investigators have had success with germline transmission of the targeted clones, providing valuable tools to the research community ( Coleman et al 2015 ; Cotton et al 2015 ). Since initial offerings of targeted ES cell clones, the KOMP project has moved forward to the production, gene expression analysis ( West et al 2015 ), and phenotyping of thousands of mouse strains ( Ring et al 2015 ), and the corresponding database management system to allow data access for researchers ( Ringwald et al 2011 ; Koscielny et al 2014 ).…”

“…The unexpected events that precluded generation of a successful allele included incorrect targeting, deletions in the 5′ end of the targeting cassette, variability of success depending on the ES cell clone initially used for targeting, and evidence for mixed populations of ES cells in the targeted “clone” ( Ryder et al 2013 ). As resource users, it is also important to consider that there are additional limitations that may arise by problematic chimera generation and/or germline transmission ( Pacholczyk et al 2008 ; Coleman et al 2015 ). While the study of Ryder et al (2013 ) characterized alleles that had been transmitted successfully, aggregate germline transmission rates appear to be around 50% [KOMP repository germline transmission rates and reference ( Cotton et al 2015 )].…”

CLEC-2 suppresses calcification in cultured osteoblasts

“…We adopted a modular strategy for the construction of targeting vectors (Supplementary 1) [29]. The podoplanin gene (Pdpn) targeting vector HTGR03003_Z_2_G05 (European Conditional Mouse Mutagenesis Program, EUCOMM) was constructed by recombineering with a modular strategy of Gateway systems, assembling the vector backbone and Pdpn from the C57BL/6J BAC libraries, which allow reporter-tagging conditional mutation of the Pdpn (EUCOMM project ID: 36335) [30].…”