Large-scale Mutagenesis Programs Research Articles

Investigating the effects of a cryptic splice site in the En2 splice acceptor sequence used in the IKMC knockout-first alleles

Targeted mouse mutants are a common tool used to investigate gene function. The International Knockout Mouse Consortium undertook a large-scale screen of mouse mutants, making use of the knockout-first allele design that contains the En2 splice acceptor sequence coupled to the lacZ reporter gene. Although the knockout-first allele was designed to interfere with splicing and thus disrupt gene function, the En2 sequence has been reported to be transcribed within the host gene mRNA due to a cryptic splice site within the En2 sequence which allows splicing to the next exon of the host gene. In some circumstances, this has the potential to permit translation of a mutant protein. Here, we describe our computational analysis of all the mouse protein-coding genes with established knockout-first embryonic stem cell lines, and our predictions of their transcription outcome should the En2 sequence be included. As part of the large-scale mutagenesis program, mutant mice underwent a broad phenotyping screen, and their phenotypes are available. No wide-scale effects on mouse phenotypes reported were found as a result of the predicted En2 insertion. However, the En2 insertion was found experimentally in the transcripts of 24 of 35 mutant alleles examined, including the five already described, two with evidence of readthrough. Splicing from the cryptic splice site also has the potential to disrupt expression of the lacZ reporter gene. It is recommended that mutant transcripts be checked for this insertion as well as for leaky transcription in studies involving knockout-first alleles.

Mutagenic sensitivity, effectiveness and efficiency of gamma rays and ethyl methane sulfonate on soft and semi-hard bread wheat (Triticum aestivum L.) varieties in the north-western Himalayan climate

Purpose The North-western Himalayan region requires unique varietal traits for the cultivation and quality of grain produced. Wheat varieties released for this zone in the past remained very popular among the farmers. However, with the passage of time certain traits such as the appearance of pathogenic rust races and grain softness have become threat to the fecundity of these genotypes and needs immediate improvement in this region. Mutation breeding facilitates improving one or two traits of a popular cultivar and to generate variability for most of plant traits upon which selection can be imposed. The purpose of this study is to evaluate the mutagenic sensitivity, effectiveness and efficiency of physical and chemical mutagens in four bread wheat varieties with differential grain texture. Materials and methods Four bread wheat varieties; HS 490, HPW 89, HPW 360 and HPW 251 were irradiated using six doses of gamma rays (γ-rays) ranging from 175 to 300 Gy; Co60 source (BARC, Mumbai, India) and six doses of ethyl methane sulfonate (EMS) ranging from 0.3 to 1.3%; EMS (Sigma-Aldrich, Bangalore, India) to assess their mutation sensitivity, effectiveness, efficiency and spectrum of induced macro-mutations in M1 and M2 generation. Results Based on mutagen sensitivity tests, both gamma rays and ethyl methane sulfonate had similar effects as the doses/concentrations increased in all four varieties. Ethyl methane sulfonate had a discernible effect on seed germination and growth parameters as compared to gamma irradiated treatments. Pollens viability studies confirmed the differential effects of both mutagens on germination and plant survivability. The LD50 and LC50 values varied between 290–315 Gy for gamma rays and 0.90–1.35% for EMS under controlled laboratory conditions, however, the range substantially differs for gamma rays (240–290 Gy) and for EMS (0.50–1.1%) under field conditions, irrespective of the variety treated. The frequency of chlorophyll mutations was low and showed a linear correlation with the doses/concentrations of the mutagen. A total of 117 putative mutants with desirable agro-morphological characteristics were also isolated. Mutagenic effectiveness and efficiency results showed that gamma irradiation doses of 250–300 Gy and ethyl methane sulfonate of 0.7–1.3% were most potent for an effective mutation breeding programme in wheat crop. Conclusions It was found that semi-hard textured varieties showed higher sensitivity to chemical mutagens as compared to soft-textured varieties. Gamma irradiation dose of 250–300 Gy and ethyl methane sulfonate concentration of 0.7–1.3% were found to be most effective and efficient across four bread wheat varieties and can be used in large scale mutagenesis programmes.

Overview of new developments in and the future of cryopreservation in the laboratory mouse

The large-scale mutagenesis programmes underway around the world are generating thousands of novel GA mouse strains that need to be securely archived. In parallel with advances in mutagenesis, the procedures used to cryopreserve mouse stocks are being continually refined in order to keep pace with demand. Moreover, the construction of extensive research infrastructures for systematic phenotyping is fuelling demand for these novel strains of mice and new approaches to the distribution of frozen and unfrozen embryos and gametes are being developed in order to reduce the dependency on the transportation of live mice. This article highlights some contemporary techniques used to archive, rederive, and transport mouse strains around the world.

A new mouse mutant for the LDL receptor identified using ENU mutagenesis

In an effort to discover new mouse models of cardiovascular disease using N-ethyl-N-nitrosourea (ENU) mutagenesis followed by high-throughput phenotyping, we have identified a new mouse mutation, C699Y, in the LDL receptor (Ldlr), named wicked high cholesterol (WHC). When WHC was compared with the widely used Ldlr knockout (KO) mouse, notable phenotypic differences between strains were observed, such as accelerated atherosclerotic lesion formation and reduced hepatosteatosis in the ENU mutant after a short exposure to an atherogenic diet. This loss-of-function mouse model carries a single base mutation in the Ldlr gene on an otherwise pure C57BL/6J (B6) genetic background, making it a useful new tool for understanding the pathophysiology of atherosclerosis and for evaluating additional genetic modifiers regulating hyperlipidemia and atherogenesis. Further investigation of genomic differences between the ENU mutant and KO strains may reveal previously unappreciated sequence functionality.

Fresh and frozen–thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl- N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen–thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl- N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen–thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability ( P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm ( P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen–thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors ( P < 0.05).

PHENOSITE: A WEB DATABASE INTEGRATING THE MOUSE PHENOTYPING PLATFORM AND THE EXPERIMENTAL PROCEDURES IN MICE

Recently, a number of collaborative large-scale mouse mutagenesis programs have been launched. These programs aim for a better understanding of the roles of all individual coding genes and the biological systems in which these genes participate. In international efforts to share phenotypic data among facilities/institutes, it is desirable to integrate information obtained from different phenotypic platforms reliably. Since the definitions of specific phenotypes often depend on a tacit understanding of concepts that tends to vary among different facilities, it is necessary to define phenotypes based on the explicit evidence of assay results. We have developed a website termed PhenoSITE (Phenome Semantics Information with Terminology of Experiments: http://www.gsc.riken.jp/Mouse/), in which we are trying to integrate phenotype-related information using an experimental-evidence-based approach. The site's features include (1) a baseline database for our phenotyping platform; (2) an ontology associating international phenotypic definitions with experimental terminologies used in our phenotyping platform; (3) a database for standardized operation procedures of the phenotyping platform; and (4) a database for mouse mutants using data produced from the large-scale mutagenesis program at RIKEN GSC. We have developed two types of integrated viewers to enhance the accessibility to mutant resource information. One viewer depicts a matrix view of the ontology-based classification and chromosomal location of each gene; the other depicts ontology-mediated integration of experimental protocols, baseline data, and mutant information. These approaches rely entirely upon experiment-based evidence, ensuring the reliability of the integrated data from different phenotyping platforms.

A genome end-game: understanding gene function in the nervous system.

The mouse has offered great promise as a model organism to study brain function and behavior; however, neurological phenotypes in mice are often detected by individual investigators in a low-throughput fashion, studying natural variants of mice or mice with spontaneous mutations or gene knockdowns. In 2000, because of the success of large-scale mutagenesis programs in other model organisms (the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans and zebrafish) and the success of large-scale mouse mutagenesis efforts in Europe (www.mgu.har.mrc.ac.uk/mutbase/; www.gsf.de/ieg/groups/enumouse.html), the National Institutes of Health (NIH) began to support three mutagenesis centers in the US. Their collective purpose was to detect, characterize and distribute new mouse mutants with primarily neurological phenotypes. These centers are located at Northwestern University (http://genome.northwestern.edu), The Jackson Laboratory (JAX; http://nmf.jax.org) and the Tennessee Mouse Genome Consortium (TMGC; www.tnmouse.org).

Towards new models of disease and physiology in the neurosciences: the role of induced and naturally occurring mutations.

There is a dearth of good mouse models for central nervous system (CNS) disorders. However, the development of gene-targeted technology and the recognition of the importance of the mouse as a model organism have led to the development of a range of behavioural tests for mice. Spontaneous mutations in mice have already provided important information about the role of novel gene products in disorders such as epilepsy and deafness. This has provided the impetus to the establishment of large-scale mutagenesis programmes to generate new mutations. Tests of sensory and motor function have previously been most frequently used as these are simple to perform and the phenotypes are relatively obvious. Subtle phenotypes, of relevance to pyschiatric disorders such as anxiety and schizophrenia, can be detected using more complex tests. Screens such as prepulse inhibition and startle have been adapted for mice and these can be run with relatively high throughput using fully automated equipment. Other behaviours such as sleep and circadian rhythms, learning and memory and nociception can also be assessed. New technological advances in non-invasive imaging and neurochemical analyses have meant that these techniques can be readily applied to mouse phenotyping. The use of these screens together with mutagenesis is already beginning to increase the numbers of mouse models of potential relevance to CNS diseases.

Current awareness on comparative and functional genomics [bibliography

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on comparative and functional genomics. Each bibliography is divided into 16 sections. 1 Reviews & symposia; 2 General; 3 Large-scale sequencing and mapping; 4 Genome evolution; 5 Comparative genomics; 6 Gene families and regulons; 7 Pharmacogenomics; 8 Large-scale mutagenesis programmes; 9 Functional complementation; 10 Transcriptomics; 11 Proteomics; 12 Protein structural genomics; 13 Metabolomics; 14 Genomic approaches to development; 15 Technological advances; 16 Bioinformatics. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted

Large numbers of mice established by in vitro fertilization with cryopreserved spermatozoa: implications and applications for genetic resource banks, mutagenesis screens, and mouse backcrosses.

Recent advances in the methodologies for cryopreservation of mouse spermatozoa have opened up a number of opportunities for mouse geneticists. We have investigated various applications for this relatively new technology and have explored the potential of sperm freezing coupled with IVF for archiving, stock building, and the rapid establishment of backcrosses. Firstly, we investigated the use of sperm freezing for the archiving of (C3H/HeH x BALB/c)F(1) progeny from a large-scale mutagenesis program. We have demonstrated that it is possible to establish efficient, comprehensive, and deep archives and that potentially thousands of offspring can be derived from the frozen spermatozoa of a single mutant male mouse. Secondly, we examined the efficacy of sperm freezing for a number of other genotypes. For at least some genotypes, frozen spermatozoa can be utilized to rapidly build stock far more quickly than by conventional methods. Finally, we demonstrated that it is feasible to use frozen spermatozoa from the mouse mutant archive for the rapid generation of mutant backcross progeny.

Engineering a mouse balancer chromosome

Balancer chromosomes are genetic reagents that are used in Drosophila melanogaster for stock maintenance and mutagenesis screens. Despite their utility, balancer chromosomes are rarely used in mice because they are difficult to generate using conventional methods. Here we describe the engineering of a mouse balancer chromosome with the Cre-loxP recombination system. The chromosome features a 24-centiMorgan (cM) inversion between Trp53 (also known as p53) and Wnt3 on mouse chromosome 11 that is recessive lethal and dominantly marked with a K14-Agouti transgene. When allelic to a wild-type chromosome, the inversion suppresses crossing over in the inversion interval, accompanied by elevated recombination in the flanking regions. The inversion functions as a balancer chromosome because it can be used to maintain a lethal mutation in the inversion interval as a self-sustaining trans-heterozygous stock. This strategy can be used to generate similar genetic reagents throughout the mouse genome. Engineering of visibly marked inversions and deficiencies is an important step toward functional analyses of the mouse genome and will facilitate large-scale mutagenesis programs.