In conclusion, the main objective of the two efforts was achieved with the generation of nearly 800 mouse models, which are available and accompanied by a broad-based phenotype analysis. Furthermore, both programmes reach similar conclusions on mutant lethality, finding that around 30 % of mouse mutants are not viable. Both programmes highlighted the efficiency of the broad-based phenotyping approaches, identifying at least one phenotype in around 80 % of the mouse mutants. The mouse clinics have provided a new set of data on gene function to the scientific community while improving the throughput and reducing the cost of the analysis. The results summarized here provide many logistical, operational, and scientific lessons that will be vital as we begin the next step in undertaking a global project of exploration of the function of genes and generation of an encyclopaedia of the mammalian genome by the IMPC.
Nevertheless, additional resources are required, most importantly the completion of the project of generating mutant ES cells for all loci in the mouse genomes: not all the genes are available yet in the IKMC. New initiatives have already begun with three new programmes—The EUCOMM-Tools for Functional Annotation of the Mouse Genome (EUCOMMTools), the Knockout Mouse Phenotyping Programme (KOMP2), and Norcommt2ls—to generate a final series of mutant ES cells, along with a new set of genetic tools for conditional genetics. Since 2011, miR knockout (KO) ES cells are also available on the IKMC web site. Prosser et al. (2011
) have developed a resource of vectors and ES cells for targeted deletion of microRNAs in mice and show how to generate, using Recombinase Mediated Cassette Exchange (RMCE) from this miR KO allele, a reporter of the miR promoter activity, the conditional KO, and how to mutate one or other miR organised within the miR clusters. Further resources will be needed for long noncoding RNAs, for point mutations, and for copy number variants, and more generally to decipher the regulatory mechanism of the transcriptional and genome organisation. In addition, it will be important to consider routes to explore heterozygous lethal or haploinsufficient mutations that escape current analyses.
In the two programmes discussed, the mutants generated so far and analysed were almost all derived from one allele, the tm1a allele. A limitation of this approach is the efficient transcription stop due to the presence of the polyA. We know from a series of experiments in the different clinics that some tm1a alleles do not lead to full expected inactivation of the targeted gene. One way to circumvent the problem is to use the tm1b allele carrying the deletion of the critical exon(s) without the removal of the lacZ sequence (or the lacZ and neo sequences in the promoterless mutants). To obtain such a tm1b allele, different universal deleters are available, but some deleters are likely to be preferred. For example, the Gt(ROSA)26Sortm1(ACTB-cre,-EGFP)Ics
deleter eliminates one generation of breeding due to the maternal accumulation of the Cre in the oocytes allowing the accumulation of the recombinase during oogenesis and the direct recovery of individuals carrying the deletion without the Cre transgene (Birling et al. 2011
). In addition, the line was developed on a pure C57BL/6 N genetic background and the presence of the transgene can be genotyped by GFP fluorescence.
The main outcomes of the first set of mutants that were analysed are the high level of homozygote lethality observed with over 30 % of the lines studied and the high level of phenotype annotations associated with heterozygous mutants. Nevertheless, a number of additional approaches should be considered. First, the lacZ pattern of the mutant allele should be explored. The use of tm1a or tm1b allele’s lacZ reporter enables us to study the expression of the gene with good sensitivity in heterozygotes. Of course, an inconvenience of this strategy is that regulatory elements or other genomic elements (e.g., ncRNA, miRNA) in the intron of the gene are modified as well. In addition, the locus might code for various isoforms with different promoters. If the isoform expressed in the organ of interest is different from the isoform targeted and if it uses a different (endogenous) promoter, the lacZ will not be expressed as expected. Second, the phenotyping pipeline should also integrate an embryonic screen to determine key steps of embryonic development. Some centres have already developed an embryonic lethal screening pipeline on a limited number of lines with some success.
The EUMODIC and SANGER-MGP programmes pioneered the high-throughput production and comprehensive analysis of mice knockouts. Considerable experience has been gained in the logistics and operation of phenotyping pipelines that will underpin future developments in international phenotyping programmes. For example, much information has been gathered on the effectiveness and sensitivity of phenotyping platforms that will prove valuable in the design of future pipelines, including the introduction of new assays to cover physiological systems. Along with the new partners of the IMPC, a new adult pipeline has been developed and the establishment of a unique and high-throughput embryonic pipeline is under discussion. As part of this process, additional assays for immune systems, inflammation, respiration, cognition, and behaviour have all been considered. In addition, building the research infrastructure capacities required in Europe for these large-scale efforts, as well as for access of individual researchers to high-throughput phenotyping, is the task of the pan-European Infrafrontier programme, which has been prioritized by ESFRI (European Strategy Forum on Research Infrastructures). Infrafrontier also secures sustainable access to mouse models archived in EMMA. Similar efforts are underway in North America and in Asia. With these new developments, we can expect that the upcoming large-scale phenotyping pipeline operated by the IMPC will prove even more effective in delivering broad-based phenotype information on a wide variety of body systems and physiological functions.