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PubMed2007

Genetic analysis of the innate immune responses in wild-derived inbred strains of mice.

The vertebrate immune system has evolved to recognize nucleic acids of bacterial and viral origin. Microbial DNA, as well as synthetic oligonucleotides based on these motifs, activates innate immune pathways mediated by the family of Toll-like receptors (TLR) initiating a cascade of signals in immune cells necessary for responses to pathogens. However, not all of the proteins that participate in TLR-mediated responses have been identified. In studies described herein, we observed significant variation in innate immune responses among selected wild-derived strains of mice. Specifically, we show that mice of MOLF/Ei, Czech/Ei, and MSM/Ms strains are hypo-responsive to polyinosinic-polycytidylic acid (poly(I:C)) because of a mutation in Tlr3. In addition, we discovered a hypo-response to cytosine guanine dinucleotide in MOLF/Ei mice and established that it is not linked to Tlr9, but to another locus. Further inquiry revealed that this hypo-response is transmitted as a monogenic dominant trait that can be mapped and cloned through positional cloning methods. These results suggest the existence of a novel molecule that can alter pro-inflammatory signals or activate additional signal transduction pathways. In addition, they support the wild-derived mouse strain as a forward genetic tool for the identification of novel immunological phenotypes.

Amino Acid Sequence↗
PubMed2005

Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system.

Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.

Alleles↗
PubMed2004

Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish.

The zebrafish is an attractive vertebrate model for genetic studies of development, apoptosis, and cancer. Here we describe a transgenic zebrafish line in which T- and B-lymphoid cells express a fusion transgene that encodes the zebrafish bcl-2 protein fused to the enhanced green fluorescence protein (EGFP). Targeting EGFP-bcl-2 to the developing thymocytes of transgenic fish resulted in a 2.5-fold increase in thymocyte numbers and a 1.8-fold increase in GFP-labeled B cells in the kidney marrow. Fluorescent microscopic analysis of living rag2-EGFP-bcl-2 transgenic fish showed that their thymocytes were resistant to irradiation- and dexamethasone-induced apoptosis, when compared with control rag2-GFP transgenic zebrafish. To test the ability of bcl-2 to block irradiation-induced apoptosis in malignant cells, we compared the responsiveness of Myc-induced leukemias with and without EGFP-bcl-2 expression in living transgenic zebrafish. T-cell leukemias induced by the rag2-EGFP-Myc transgene were ablated by irradiation, whereas leukemias in double transgenic fish expressing both Myc and EGFP-bcl-2 were resistant to irradiation-induced apoptotic cell death. The forward genetic capacity of the zebrafish model system and the ability to monitor GFP-positive thymocytes in vivo make this an ideal transgenic line for modifier screens designed to identify genetic mutations or small molecules that modify bcl-2-mediated antiapoptotic pathways.

Amino Acid Sequence↗
PubMed2003

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis.

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.

Animals↗
PubMed2025

A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis.

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in Chlamydia trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase the overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.IMPORTANCEChlamydia trachomatis is a prevalent human pathogen exerting a tremendous negative impact on human health. A complete understanding of how these bacteria create and maintain an intracellular niche and avoid/subvert host defense mechanisms to cause disease is lacking. The utility of transposon-mediated, random mutagenesis in supporting forward genetic studies is well established in a multitude of genetically tractable systems. This study reports the development of a plasmid-based system capable of generating mutant pools and supporting subsequent isolation of individual transposon mutants. This step is an important advance in providing a mechanism capable of supporting downstream studies interrogating chlamydial biology.

Chlamydia trachomatis↗
PubMed2005

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Animals↗
PubMed2003

Expressing functional siRNAs in mammalian cells using convergent transcription.

BACKGROUND: The use of small interfering RNAs (siRNAs) as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs) or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi)-mediated gene silencing. RESULTS: With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. CONCLUSION: The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences. Furthermore, the cassette design reported is ideal for both routine use in controlling specific gene expression and construction of randomised RNAi expression libraries for use in unbiased forward genetic selections.

Cell Line↗
PubMed2003

Molecular components of the endoderm specification pathway in Xenopus tropicalis.

Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate endoderm specification. However, loss-of-function analysis is required to resolve the precise function of the genes involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17alpha, tSox17beta, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes, thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better probe penetration than in laevis whole-mount in situ hybridizations-allowing us to visualize transcripts in the deep endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large number of tropicalis-expressed sequence tags that are now available.

Animals↗
PubMed2004

Identification of recessive maternal-effect mutations in the zebrafish using a gynogenesis-based method.

In animal species, early developmental processes are driven by maternally derived factors. Here, we describe a forward genetics approach to identify recessive mutations in genes encoding such maternal factors in the zebrafish. We used a gynogenesis-based approach to identify 14 recessive maternal-effect mutations. Homozygosity for these mutations in adult females leads to the inviability of their offspring. Confocal microscopy of embryos labeled with a DNA dye and a membrane marker allowed us to further analyze mutant embryos for defects in nuclear and cellular divisions. The mutations result in a range of defects in early developmental processes, including egg activation, early nuclear events, mitosis, cytokinesis, axial patterning, and gastrulation. Our effort constitutes a systematic attempt to identify maternal-effect genes in a vertebrate species. The sample of mutations that we have identified reflects the diversity of maternally driven functions in early development and underscores the importance of maternal factors in this process.

Animals↗
PubMed2001

Transposition and gene disruption in the male germline of the mouse.

We have tested a synthetic, functional, transposon called Sleeping Beauty for use in mice as a germline insertional mutagen. We describe experiments in which mutagenic, polyadenylation-site trapping, transposon vectors were introduced into the germline of mice. When doubly transgenic males, expressing the Sleeping Beauty transposase gene (SB10) and harboring poly(A)-trap transposon vectors, were outcrossed to wild-type females, offspring were generated with new transposon insertions. The frequency of new transposon insertion is roughly two per male gamete. These new insertions can be passed through the germline to the next generation and can insert into or near genes. We have generated a preliminary library of 24 mice harboring 56 novel insertion sites, including one insertion into a gene represented in the EST database and one in the promoter of the galactokinase (Gck) gene. This technique has promise as a new strategy for forward genetic screens in the mouse or functional genomics.

Animals↗
PubMed2004

Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8.

A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells. Homozygous mutant mice are sterile. Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity. Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I. Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8. Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms. However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements.

Animals↗
PubMed2000

CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans.

Forward genetic screens using novel assays of nematode chemotaxis to soluble compounds identified three independent transposon-insertion mutations in the gene encoding the Caenorhabditis elegans dynein heavy chain (DHC) 1b isoform. These disruptions were mapped and cloned using a newly developed PCR-based transposon display. The mutations were demonstrated to be allelic to the che-3 genetic locus. This isoform of dynein shows temporally and spatially restricted expression in ciliated sensory neurons, and mutants show progressive developmental defects of the chemosensory cilia. These results are consistent with a role for this motor protein in the process of intraflagellar transport; DHC 1b acts in concert with a number of other proteins to establish and maintain the structural integrity of the ciliated sensory endings in C. elegans.

Amino Acid Sequence↗
PubMed2001

Specification and morphogenesis of the zebrafish larval head skeleton.

Forward genetic analyses can reveal important developmental regulatory genes and how they function to pattern morphology. This is because a mutated gene can produce a novel, sometimes beautiful, phenotype that, like the normal phenotype, immediately seems worth understanding. Generally the loss-of-function mutant phenotype is simplified from the wild-type one, and often the nature of the pattern simplification allows one to deduce how the wild-type gene contributes to patterning the normal, more complex, morphology. This truism seems no less valid for the vertebrate head skeleton than for other and simpler cases of patterning in multicellular plants and animals. To show this, we review selected zebrafish craniofacial mutants. "Midline group" mutations, in genes functioning in one of at least three signal transduction pathways, lead to neurocranial pattern truncations that are primarily along the mediolateral axis. Mutation of lazarus/pbx4, encoding a hox gene partner, and mutation of valentino/kreisler, a hox gene regulator, produce anterior-posterior axis disruptions of pharyngeal cartilages. Dorsoventral axis patterning of the same cartilages is disrupted in sucker/endothelin-1 mutants. We infer that different signal transduction pathways pattern cartilage development along these three separate axes. Patterning of at least the anterior-posterior and dorsoventral axes have been broadly conserved, e.g., reduced Endothelin-1 signaling similarly perturbs cartilage specification in chick, mouse, and zebrafish. We hypothesize that Endothelin-1 also is an upstream organizer of the patterns of cellular interactions during cartilage morphogenesis.

Animals↗
PubMed2004

The path in fungal plant pathogenicity: many opportunities to outwit the intruders?

The number of genes implicated in the infection and disease processes of phytopathogenic fungi is increasing rapidly. Forward genetic approaches have identified mutated genes that affect pathogenicity, host range, virulence and general fitness. Likewise, candidate gene approaches have been used to identify genes of interest based on homology and recently through 'comparative genomic approaches' through analysis of large EST databases and whole genome sequences. It is becoming clear that many genes of the fungal genome will be involved in the pathogen-host interaction in its broadest sense, affecting pathogenicity and the disease process in planta. By utilizing the information obtained through these studies, plants may be bred or engineered for effective disease resistance. That is, by trying to disable pathogens by hitting them where it counts.

Fungi↗
PubMed1999

Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating.

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs. Feminization of parts of the antennal lobe and mushroom body by targeted expression of a female-determining gene transformer+ (tra+) drives the male to court other males. Mutations in the tra target gene fruitless (fru), which is expressed in the antennal lobe as well as the suboesophageal ganglion (the gustatory inputs are processed here), also induce homosexual courtship in males. These results suggest that sensory inputs mediated and/or processed by the tarsal receptors, suboesophageal ganglion, antennal lobe and mushroom body contribute to the regulation of male-female courtship. Mosaic analysis localized the neural center for male courtship behavior to the posterior dorsal brain, in which the sensory information processed by the aforementioned neural structures may be integrated. Another mosaic study mapped the neural center for female sexual behavior, as measured by her receptiveness to copulation, to the anterior dorsal brain. The issue as to how the mutations that reduce female sexual receptiveness, e.g. dissatisfaction (dsf), spinster (spin) and chaste (cht), affect the structure and/or function of this neural center deserves to be addressed urgently.

Animals↗
PubMed2000

Tests of the mouse visual system.

To apply the approach of forward genetics (e.g., gene identification with mutagenesis and screening, followed by positional cloning) to the mouse, it is necessary to have available screening tests that can be applied rapidly to individual mice and that give a reliable assessment of visual function. This paper reviews the strengths and limitations of two anatomical tests related to visual function, fundus examination and retinal histological examination. Two tests that do not depend on behavior of a conscious animal are reviewed: the electroretinogram and the visual evoked potentials of the cortex. Eight behavioral tests are also summarized: maze-based tests, cued fear conditioning, tests based on conditioned suppression, visual placing, optokinetic nystagmus, pupillary reflex, and light-induced shifts in circadian phase. It is recommended that retinal histology, the electroretinogram, and visual-evoked potentials be used at the present time for screening because they assess the function and structure of the visual system rapidly and reliably. In fact, the electroretinogram (or visually evoked potentials) can be recorded from several animals simultaneously in response to the same stimulus. It is also recommended that efforts be made to develop more appropriate, automated, behavioral tests of visual perception than are now available, particularly tests that rely solely on rewarding visually evoked behavior. Two other promising behavioral tests are cued fear conditioning and variants of maze tests.

Animals↗
PubMed2005

Medaka double mutants for color interfere and leucophore free: characterization of the xanthophore-somatolactin relationship using the leucophore free gene.

Somatolactin (SL) plays an essential role in body-color regulation in medaka and is encoded by the color interfere (ci) locus. The ci mutant fish possess constitutively increased numbers of leucophores and a concomitant decrease in visible xanthophores. However, the mechanism of action of SL on these cell types, and the role of SL in body-color regulation in other species, is unknown. In this study, we verified an SL-xanthophore relationship in ci mutant fish using the leucophore free (lf) gene. Histological observation of lf larvae indicated that these mutants do not possess differentiated leucophores. The ci-lf double mutant, whose genotype was confirmed using DNA markers, lacked leucophores; however, the number of xanthophores remained low, demonstrating that leucophores are not necessary for mediating SL signaling to xanthophores. This finding suggests a conserved function for SL in xanthophore regulation across species, rather than the evolution of a medaka-specific and leucophore-dependent role of SL in body-color regulation. Our results also demonstrate that the lf gene has an indispensable role in leucophore development epistatic to SL signaling. The lf gene has not been cloned. The high-resolution recombination map surrounding the lf locus constructed in this study, together with medaka whole genome sequences that will be released soon, will allow the rapid cloning of the lf gene by forward genetic approaches.

Animals↗
PubMed2004

Implementing large-scale ENU mutagenesis screens in North America.

A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening.

Animals↗