Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs Syst Biol Med
Impact Factor: 3.542

Large‐scale mouse knockouts and phenotypes

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Abstract Standardized phenotypic analysis of mutant forms of every gene in the mouse genome will provide fundamental insights into mammalian gene function and advance human and animal health. The availability of the human and mouse genome sequences, the development of embryonic stem cell mutagenesis technology, the standardization of phenotypic analysis pipelines, and the paradigm‐shifting industrialization of these processes have made this a realistic and achievable goal. The size of this enterprise will require global coordination to ensure economies of scale in both the generation and primary phenotypic analysis of the mutant strains, and to minimize unnecessary duplication of effort. To provide more depth to the functional annotation of the genome, effective mechanisms will also need to be developed to disseminate the information and resources produced to the wider community. Better models of disease, potential new drug targets with novel mechanisms of action, and completely unsuspected genotype–phenotype relationships covering broad aspects of biology will become apparent. To reach these goals, solutions to challenges in mouse production and distribution, as well as development of novel, ever more powerful phenotypic analysis modalities will be necessary. It is a challenging and exciting time to work in mouse genetics. WIREs Syst Biol Med 2012, 4:547–563. doi: 10.1002/wsbm.1183 This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Mammalian Physiology in Health and Disease Models of Systems Properties and Processes > Organismal Models Physiology > Physiology of Model Organisms

This WIREs title offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images


Common alleles in embryonic stem cell (ESC) resources in the C57BL/6N genetic background. (a) The TIGM allele incorporates a retroviral insertion carrying a splice acceptor (SA) site upstream of a selectable cassette (βgeo) and a poly‐adenylation (pA) signal; retroviral long terminal repeats (LTR) = black boxes, mutated gene exons = red boxes. (b) the EUCOMM/KOMP tm1a allele includes LoxP sites (green arrowheads) around a critical exon (CE, brown box) of the target gene. The upstream intron includes a mutagenic unit composed of a SA‐β‐galactosidase‐pA (βgal) and neomycin phospho‐transferase (neo) drug‐resistance cassettes for expression analysis and clonal selection respectively. These cassettes are surrounded by FRT sites (yellow arrowheads) for FLP recombinase. As both cassettes carry polyA signals, the tm1a allele is expected to disrupt the target gene expression by interrupting transcription. The tm1a allele can be modified in ESC or in mice, by expression of Cre recombinase to produce the tm1b allele, which is expected to disrupt the target gene by deletion of the CE in addition to the effect on transcription. The tm1a allele can also be converted to tm1c by expression of the FLP recombinase. The tm1c allele is a conditional allele which is not expected to affect the gene's expression but can serve as excellent substrate for time and/or tissue‐specific mutagenesis by expression of Cre which converts it to the tm1d allele.

[ Normal View | Magnified View ]

Examples of phenodeviants identified by the Sanger Mouse Genetics Project primary phenotyping screen. (a) Sparse hair appeared in a wave‐like pattern on the ventral side of Krt76 −/− mice (n = 7/7 males, 4/4 females) compared with matched controls (b). Furthermore, pigmented footpads were observed in all Krt76−/− mice assessed (c), and this abnormal pigmentation closely mirrored strong and specific lacZ reporter gene expression in Krt76+/− animals (d). Expression analysis using the lacZ reporter gene also revealed a strong but distinct Ctnnal1 expression pattern in the peripheral nervous system including (e), intercostal nerves and (f), enteric nerves within the large intestine, noteworthy because of its candidacy for Hirschsprung disease, a congenital disorder affecting innervation of the colon. Consistent with its association with Meckel syndrome type 1, 67% of E14.5 Mks1−/− embryos presented with (g), polydactyly, eye defects, and edema and (h), craniofacial abnormalities and hemorrhaging (indicated by arrows). (i) Fourteen‐week‐old Zc3hc1−/− mice (n = 3/4 males, 6/7 females) presented with the absence of one rib set and associated thoracic vertebrae (T13) (indicated by arrows) compared with matched controls which progress from T13 to lumbar vertebra 1 (L1) (j).

[ Normal View | Magnified View ]

Distribution of mouse strains from the Sanger Mouse Genetics Project (MGP). In addition to the depositing strains at the European Mutant Mouse Archive and Knock Out Mouse Project repositories for long‐term distribution, the MGP distributes a large number of strains directly to the scientific community. The numbers reflect approximately 3 years of activity.

[ Normal View | Magnified View ]

Quality control for large‐scale mouse production and phenotypic analysis. Different centers perform different subsets of quality control procedures. It is important to perform them at the embryonic stem cell stage (pre‐microinjection), in the F1 heterozygous stage, and after phenotypic analysis. Commonly used tests include Southern blot, long‐range polymerase chain reaction (LRPCR), karyotyping, PCR check of the integrity of the mutagenic cassette, and demonstration of the loss of the wild‐type allele by a universal quantitative PCR (qPCR) test. Once these tests have passed in the F1 heterozygous mice, further genotyping is carried out with methods amenable to high‐throughput such as short‐range PCR (srPCR) or universal qPCR reactions that count the number of copies of the neo selection cassette or LacZ gene.

[ Normal View | Magnified View ]

Common Coat Color Segregation Schemes in Progeny Derived from Chimaera Generated with EUCOMM/KOMP ES Cells

[ Normal View | Magnified View ]

Related Articles

The mammalian phenotype ontology: enabling robust annotation and comparative analysis

Browse by Topic

Physiology > Physiology of Model Organisms
Physiology > Mammalian Physiology in Health and Disease
Models of Systems Properties and Processes > Organismal Models
Laboratory Methods and Technologies > Genetic/Genomic Methods

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts