Utilising the resources of the International Knockout Mouse Consortium: the Australian experience

Abstract

Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service.

Regulation of mitochondrial metabolism in murine skeletal muscle by the medium‐chain fatty acid receptor Gpr84

“…Mice with global deletion of Gpr84 (on a C57BL/6 background) were generated. Gpr84 tm1(KOMP)Vlcg targeted embryonic stem cells were obtained from the Knockout Mouse Project (KOMP) repository (University of California‐Davis, Davis, CA, USA), and Gpr84 heterozygous mice were produced, using these cells, by the Australian Phenomics Network (Monash node) (30). Mice were crossed with mice expressing ROSA‐Cre to remove the selection cassette, and the resulting Gpr84 heterozygous pairs were used for subsequent breeding.…”

Mutations in the Katnb1 gene cause left–right asymmetry and heart defects

“…The Katnb1 knockout mouse line was generated at the Australian Phenomics Network (APN) Monash University Node using a DEVELOPMENTAL DYNAMICS Taily 2 7 28.9 Katnb1 1/Taily Taily 2 7 28.9 Katnb1 1/Taily EUCOMM KO-first condition ready ES clone (HEPD0636_3_G09) using standard methods (Cotton et al, 2015). To disrupt the Katnb1 gene (ENSMUSG00000031787), the FRT-LacZ-loxP-Neo-FRT-loxP-Katnb1-exon4-loxP cassette was inserted in intron 3 of the Katnb1 gene (Katnb1 tm1a(EUCOMM)Hmgu allele).…”

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

“…Based upon past experience and anecdotal evidence that germline transmission may be more difficult to achieve with C57BL/6 ES cells, ES2M staff recommended four ES cell clones per GOI be purchased from EUCOMM ( 35 ). For GOI-1, we found that only one of the four clones passed quality control (two clones had an abnormal karyotype and one clone did not actually contain the tm1a allele).…”

“…For GOI-2, only three of four clones were available for shipping and one clone had ≤50% euploidy and therefore failed the necessary criteria for injection ( 35 ). GOI-2 clone #3 was first chosen for microinjection because of its favorable karyotype but failed to produce live births from either injection round.…”

“…On average, the time from ES2M order submission to arrival of heterozygous tm1a mice at our animal facility was 28 months. It is important for researchers to accommodate at least 4 months from the time of order for receipt of the ES cells from EUCOMM, a common processing time for the international consortia ( 35 ). In our case, we experienced a longer time to germline transmission than most because of the quality of our ES cells – while KOMP-derived ES cells are routinely subjected to quality control screening before dispatch, this has only recently been implemented by EUCOMM ( 35 ).…”