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PubMed2005

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.

Alkylating Agents↗

PubMed1999

[The molecular basis of primary immunodeficiency syndrome and the current therapeutic approach for the disease].

The development of genetic tools in recent years has enabled characterization of the molecular basis of several genetic disorders of the human system. These findings have led to better understanding of the physiology of the human system as well as to the establishment of improved diagnosis tools offering new perspectives in genetic counseling and therapeutics. Recently, genetic disorders of the regulation of the immune system have been described both in humans and mice. These diseases are characterized by defective control of activation/proliferation of lymphocytes and macrophages. These findings opened a new field in immunological research. Detailed analysis of the molecular basis and functional characterization of the affected gene product may help to understand the phenotype of the patients. Considerable progress has been made in the therapeutics for these patients including unrelated bone marrow transplantation, cord blood stem cell transplantation, and gene therapy. Ex vivo expanded activated T cell transfusion, a new therapeutic approach to control opportunistic infection, may be effective for some patients with impaired T cell function.

Animals↗

PubMed2000

Unnatural ligands for engineered proteins: new tools for chemical genetics.

Small molecules that modulate the activity of biological signaling molecules can be powerful probes of signal transduction pathways. Highly specific molecules with high affinity are difficult to identify because of the conserved nature of many protein active sites. A newly developed approach to discovery of such small molecules that relies on protein engineering and chemical synthesis has yielded powerful tools for the study of a wide variety of proteins involved in signal transduction (G-proteins, protein kinases, 7-transmembrane receptors, nuclear hormone receptors, and others). Such chemical genetic tools combine the advantages of traditional genetics and the unparalleled temporal control over protein function afforded by small molecule inhibitors/activators that act at diffusion controlled rates with targets.

Alleles↗

PubMed1996

The degree of lateralization of paw usage (handedness) in the mouse is defined by three major phenotypes.

Lateralization of paw usage in the laboratory mouse may be a useful model system in which to assess the genetic and developmental cause of asymmetry of hand usage. With a set number of paw reaches from a centrally placed food tube, individual mice from an inbred strain will exhibit a reliable number of left and right paw reaches. For a single inbred strain, there are approximately equal numbers of left-pawed and right-pawed mice, but strain differences have been reported in the degree of lateralization of paw preference. We reported a preliminary strain survey in which the strains appeared to fall into two groups of highly lateralized and weakly lateralized paw preference (Biddle et al., 1993). We review here our expanded survey of genetically different strains and stocks of the laboratory mouse, including different species and subspecies. The major genetic trait is the degree of lateralization of paw preference and the strain differences appear to fall into three major classes of highly lateralized, weakly lateralized, and ambilateral preference. The trait exhibits both additivity and dominance in preliminary reciprocal crosses, depending on which strain pairs are used. The wide difference between strains that have highly lateralized and ambilateral paw preference suggests specific genetic tools that could be used to begin a genetic dissection of the causes of this trait. Preliminary assessment of the size of the corpus callosum in three strains with significantly different degrees of lateralization suggests that genetically determined deficiencies and absence of this structure are not the direct cause of the strain differences in the trait of degree of lateralization. In the expanded survey, some strains appear to exhibit a directional deviation from equal numbers of mice with left and right paw usage. Therefore, direction of paw usage may not be a genetically neutral trait, but replicate assessments and genetic tests are needed to confirm this.

Animals↗

PubMed2003

Overlapping deletions spanning the proximal two-thirds of the mouse t complex.

Chromosome deletion complexes in model organisms serve as valuable genetic tools for the functional and physical annotation of complex genomes. Among their many roles, deletions can serve as mapping tools for simple or quantitative trait loci (QTLs), genetic reagents for regional mutagenesis experiments, and, in the case of mice, models of human contiguous gene deletion syndromes. Deletions also are uniquely suited for identifying regions of the genome containing haploinsufficient or imprinted loci. Here we describe the creation of new deletions at the proximal end of mouse Chromosome (Chr) 17 by using the technique of ES cell irradiation and the extensive molecular characterization of these and previously isolated deletions that, in total, cover much of the mouse t complex. The deletions are arranged in five overlapping complexes that collectively span about 25 Mbp. Furthermore, we have integrated each of the deletion complexes with physical data from public and private mouse genome sequences, and our own genetic data, to resolve some discrepancies. These deletions will be useful for characterizing several phenomena related to the t complex and t haplotypes, including transmission ratio distortion, male infertility, and the collection of t haplotype embryonic lethal mutations. The deletions will also be useful for mapping other loci of interest on proximal Chr 17, including T-associated sex reversal ( Tas) and head-tilt ( het). The new deletions have thus far been used to localize the recently identified t haplolethal ( Thl1) locus to an approximately 1.3-Mbp interval.

Animals↗

PubMed2003

Integrative physiology and functional genomics of epithelial function in a genetic model organism.

Classically, biologists try to understand their complex systems by simplifying them to a level where the problem is tractable, typically moving from whole animal and organ-level biology to the immensely powerful "cellular" and "molecular" approaches. However, the limitations of this reductionist approach are becoming apparent, leading to calls for a new, "integrative" physiology. Rather than use the term as a rallying cry for classical organismal physiology, we have defined it as the study of how gene products integrate into the function of whole tissues and intact organisms. From this viewpoint, the convergence between integrative physiology and functional genomics becomes clear; both seek to understand gene function in an organismal context, and both draw heavily on transgenics and genetics in genetic models to achieve their goal. This convergence between historically divergent fields provides powerful leverage to those physiologists who can phrase their research questions in a particular way. In particular, the use of appropriate genetic model organisms provides a wealth of technologies (of which microarrays and knock-outs are but two) that allow a new precision in physiological analysis. We illustrate this approach with an epithelial model system, the Malpighian (renal) tubule of Drosophila melanogaster. With the use of the beautiful genetic tools and extensive genomic resources characteristic of this genetic model, it has been possible to gain unique insights into the structure, function, and control of epithelia.

Animals↗

PubMed1998

A rapid method for disrupting genes in the Escherichia coli genome.

The entire genomic sequence of Escherichia coli has recently been completed. To gain insight into the function of the vast array of yet uncharacterized open reading frames (ORFs), a variety of new genetic tools will be required. Here we examined a genetic system, using an integration plasmid vector (named pINT007), for rapid construction of disruption mutants of any ORF in E. coli. It was found that the vector allows us to rapidly construct a disruption mutant for any gene on the chromosome as a cointegrate, furthermore, resolution of the resulting cointegrate can be surely accomplished by using a pair of the bla (ampicillin resistant) genes on the vector as a positive-selection marker.

Escherichia coli↗

PubMed1997

[Genetic anomalies of the human immune system].

The development of genetic tools in the recent years has made possible the characterization of the molecular basis from several genetic disorders of the human system. These findings lead to a better understanding of the physiology of the human system as well as to the definition of improved diagnosis tools offering new perspectives in genetic counseling and therapeutics. Recently, genetic disorders of the regulation of the immune system have been described both in men and mice. These diseases are characterized by a defective control of activation/proliferation of lymphocytes and macrophages. Their study opens a new field in immunological research. Understanding of their molecular basis showed help to define susceptibility factors to autoimmune disorders.

Animals↗

PubMed2006

Tn5 transposition: a molecular tool for studying protein structure-function.

Transposon-based technologies are important genetic tools for global genome analysis and, as discussed in the present paper, in detailed studies of protein structure-function. Various different transposition systems can be used in these studies but this paper uses Tn5-related systems as a model. In particular, the following four different technologies are described in this paper: (i) using transposition to generate nested deletion families, (ii) using transposons to generate functional protein fusions to reporter functions, (iii) mapping protein secondary structures through the generation and analysis of in-frame linker insertions and (iv) using sequential transposition events to generate random gene fusions. The success of these forward genetic technologies requires that the transposition system be efficient and manifest near-random target sequence selection.

Gene Deletion↗

PubMed1995

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 1 to 6.

We typed 147 simple sequence length polymorphisms in the SWXJ recombinant inbred (RI) strain set spanning Chromosomes (Chrs) 1-6. The strain distribution pattern for these loci was combined with data from 18 previously typed loci for SWXJ, resulting in new chromosome maps for this RI set, with an average density of 3.5 cM between loci. This is the first systematic effort to develop a more highly resolved genetic map for the SWXJ RI set and thereby improves the usefulness of this genetic tool for mapping genes underlying both simple and complex genetic disorders.

Animals↗

PubMedDate not supplied

Development of novel microsatellite DNA markers from the Pacific oyster Crassostrea gigas.

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2-20; observed heterozygosity, 0.119-0.925; unbiased expected heterozygosity, 0.139-0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.

Animals↗

PubMed2000

Genetic dissection of nicotine-related behaviour: a review of animal studies.

Nicotine has a broad spectrum of behavioural effects. A considerable body of data has emerged indicating genetic factors regulate the behavioural effects of nicotine. Experimental genetic techniques have been invaluable in generating knowledge on the interrelationship of genetic factors and behavioural responsiveness to nicotine. Three different approaches have been invoked to explore the relationship of genetic factors to response to nicotine. Firstly, the classical genetic tool of inbred lines has been exploited to delineate genetic influences in the effects of nicotine. Secondly, the use of selectively bred lines has been profitably employed to reveal genetic differences in behavioural responses, such as cognition and exploration, to nicotine. These approaches have also provided useful information on the contribution of genetic factors influencing nicotinic receptors function. Finally the molecular genetic technique of gene targetting to create mice with null mutations of specific genes in the central nervous system, which is having a tremendous impact in drug addiction research, has also been employed to gain insight into the molecular and cellular basis of nicotine action. These techniques are proving to be invaluable in dissecting the role of different subunits of the nicotinic acetylcholine receptors on behaviour. This paper provides a survey of the animal studies that have used the above mentioned techniques to gain insight into genetic basis of the behavioural effects of nicotine.

Animals↗

PubMed2004

Genetic basis of sexual isolation in Drosophila melanogaster.

Sexual isolation between Zimbabwe (abbreviated as Z) and cosmopolitan (abbreviated as M) races exists in Drosophila melanogaster. Typically, when given a choice, the females from the Zimbabwe race mate only with males from the same race, whereas females from the cosmopolitan race mate readily with males from both races non-discriminatorily. Genetic tools available in this experimental organism permit the detail genetic analyses of the sexual isolation behavior. On the other hand, the search for the actual signaling systems involved in the mate recognition is still limited in this system. In this paper, we review the studies, which attempt to dissect the genetic basis of the sexual isolation system, and document the complex features of the genetic architecture and the behavioral traits that evolved at an incipient stage of speciation. The evolution and the maintenance of this behavioral polymorphism are also discussed.

Alleles↗

PubMed2026

Methods for single-pair Ascaridia galli genetic crosses.

Ascarid parasites infect a wide range of hosts, causing significant clinical and economic impacts. However, genetic tools for studying ascarid biology remain limited. We optimized genetic crosses using Ascaridia galli , a common ascarid of chickens. Sexually immature larval parasites were recovered from donors, transferred to gelatin capsules, and then given orally to recipients. We successfully established single-pair matings in 32% of crossing attempts. This method to control genetic crosses further establishes the avian model for ascarid research and will enable future studies to create a high-quality reference genome, inbreed anthelmintic resistant and sensitive lines, and investigate host-pathogen interactions.

Journal Article↗

PubMed2004

Beta-globin-gene haplotypes, mitochondrial DNA, the Y-chromosome: their impact on the genetic epidemiology of the major structural hemoglobinopathies.

The history of the discovery of the globin beta-like globin-gene haplotypes and their importance in the understanding of hemoglobinopathies has been reviewed recently. We will add in this review more recent findings and other molecular genetic tools that can help in the understanding of the genetic epidemiology of structural hemoglobinopathies, leaving the thalassemias for a later effort.

Chromosomes, Human, Y↗

PubMed2002

The thankless task of playing genetics with mammalian mitochondrial DNA: a 30-year review.

The advances obtained through the genetic tools available in yeast for studying the oxidative phosphorylation (OXPHOS) biogenesis and in particular the role of the mtDNA encoded genes, strongly contrast with the very limited benefits that similar approaches have generated for the study of mammalian mtDNA. Here we review the use of the genetic manipulation in mammalian mtDNA, its difficulty and the main types of mutants accumulated in the past 30 years and the information derived from them. We also point out the need for a substantial improvement in this field in order to obtain new tools for functional genetic studies and for the generation of animal models of mtDNA-linked diseases.

Journal Article↗

PubMed2000

Viral oncogenes as tools to study replicative senescence of human cells.

Human aging is correlated with reduced proliferation of various cell types, a phenomenon that can be reproduced in in vitro models of replicative senescence. We study senescence of several human primary cell types by analysis of age-related changes in gene expression and gene function. In a second approach, my group uses immortalizing oncogenes derived from DNA tumor viruses as genetic tools to study genetic and biochemical mechanisms underlying the progression of cells into senescence. Specifically, our work is guided by the hypothesis that cellular proteins binding to the E7 gene product of human papillomavirus are good candidates for senescence-inducing cellular factors. For several of these cellular factors, e.g. the inhibitor of cyclin-dependent kinases p21(WAF-1), a functional role in senescence has already been demonstrated.

Carbohydrate Metabolism↗

PubMed2003

Strategies for the identification of virulence determinants in human pathogenic fungi.

The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.

Fungi↗