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At least 73 records · Page 4Linked to original sources
PubMed2005

Cancer gene discovery using the Sleeping Beauty transposon.

Epidemiological and molecular data support the hypothesis that cancer results from a series of acquired somatic mutations. Discovering the initial mutations required for oncogenesis has long been a goal of cancer research. To date, the majority of causative mutations have been identified based on their ability to act in a dominant fashion and/or because they are activated by chromosomal translocations. Forward genetic screens are necessary for unbiased discovery of the remaining unknown oncogenic mutations. Two recent projects have demonstrated the feasibility of using the Sleeping Beauty transposon as an insertional mutagen for cancer gene discovery. In this article we discuss the history of cancer gene discovery and propose novel forward genetic screens using Sleeping Beauty transposon aimed at specific tissues and accelerating the discovery of recessive tumor suppressor genes.

Genes, Neoplasm↗
PubMed2005

Unraveling innate immunity using large scale N-ethyl-N-nitrosourea mutagenesis.

With the mouse genome almost entirely sequenced and readily accessible to all who wish to examine it, the challenge across most biological disciplines now lies in the decipherment of gene and protein function rather than in the realm of gene identification per se. In the field of innate immunity, forward genetic methods have repeatedly been applied to identify key sensors, adapters, and effector molecules. However, most spontaneous mutations that affect innate immune function have been mapped and cloned, and the need for new monogenic phenotypes has been felt evermore keenly. N-Ethyl-N-nitrosourea (ENU) mutagenesis is an efficient tool for the creation of aberrant monogenic innate immune response phenotypes. In this review, we will discuss the potential of the forward genetic approach and ENU mutagenesis to identify new genes and new functions of known genes related to innate immunity.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development.

BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

Animals↗
PubMed2005

Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans,Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.

Journal Article↗
PubMed2002

Techniques and probes for the study of Xenopus tropicalis development.

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis--alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis.

Animals↗
PubMed2004

Genomewide two-generation screens for recessive mutations by ES cell mutagenesis.

Forward genetic mutation screens in mice are typically begun by mutagenizing the germline of male mice with N-ethyl-N-nitrosourea (ENU). Genomewide recessive mutations transmitted by these males can be rendered homozygous after three generations of breeding, at which time phenotype screens can be performed. An alternative strategy for randomly mutagenizing the mouse genome is by chemical treatment of embryonic stem (ES) cells. Here we demonstrate the feasibility of performing genome-wide mutation screens with only two generations of breeding. Mice potentially homozygous for mutations were obtained by crossing chimeras derived from ethylmethane sulfonate (EMS)-mutagenized ES cells to their daughters, or by intercrossing offspring of chimeras. This strategy was possible because chimeras transmit variations of the same mutagenized diploid genome, whereas ENU-treated males transmit numerous unrelated genomes. This also results in a doubling of screenable mutations in a pedigree compared to germline ENU mutagenesis. Coupled with the flexibility to treat ES cells with a variety of potent mutagens and the ease of producing distributable, quality-controlled, long-term supplies of cells in a single experiment, this strategy offers a number of advantages for conducting forward genetic screens in mice.

Animals↗
PubMed2005

Discovery genetics: serendipity in basic research.

The role of serendipity in science has no better example than the discovery of spontaneous mutations that leads to new mouse models for research. The approach of finding phenotypes and then carrying out genetic analysis is called forward genetics. Serendipity is a key component of discovering and developing mice with spontaneous mutations into animal models of human disease. In this article, the role of serendipity in discovering and developing mouse models is described within a program at The Jackson Laboratory that capitalizes on serendipitous discoveries in large breeding colonies. Also described is how any scientists working with mice can take advantage of serendipitous discoveries as a research strategy to develop new models. Spontaneous mutations cannot be planned but happen in all research mouse colonies and are discovered as unexpected phenotypes. The alert scientist or technician can rationally exploit such chance observations to create new research opportunities.

Animals↗
PubMed2022

Flavivirus-Host Interaction Landscape Visualized through Genome-Wide CRISPR Screens.

Flaviviruses comprise several important human pathogens which cause significant morbidity and mortality worldwide. Like any other virus, they are obligate intracellular parasites. Therefore, studying the host cellular factors that promote or restrict their replication and pathogenesis becomes vital. Since inhibiting the host dependency factors or activating the host restriction factors can suppress the viral replication and propagation in the cell, identifying them reveals potential targets for antiviral therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has provided an effective means of producing customizable genetic modifications and performing forward genetic screens in a broad spectrum of cell types and organisms. The ease, rapidity, and high reproducibility of CRISPR technology have made it an excellent tool for carrying out genome-wide screens to identify and characterize viral host dependency factors systematically. Here, we review the insights from various Genome-wide CRISPR screens that have advanced our understanding of Flavivirus-Host interactions.

Humans↗
PubMed2002

Genetic complementation in apicomplexan parasites.

A robust forward genetic model for Apicomplexa could greatly enhance functional analysis of genes in these important protozoan pathogens. We have developed and successfully tested a genetic complementation strategy based on genomic insertion in Toxoplasma gondii. Adapting recombination cloning to genomic DNA, we show that complementing sequences can be shuttled between parasite genome and bacterial plasmid, providing an efficient tool for the recovery and functional assessment of candidate genes. We show complementation, gene cloning, and biological verification with a mutant parasite lacking hypoxanthine-xanthine-guanine phosphoribosyltransferase and a T. gondii cDNA library. We also explored the utility of this approach to clone genes based on function from other apicomplexan parasites using Toxoplasma as a surrogate. A heterologous library containing Cryptosporidium parvum genomic DNA was generated, and we identified a C. parvum gene coding for inosine 5-monophosphate-dehydrogenase (IMPDH). Interestingly, phylogenetic analysis demonstrates a clear eubacterial origin of this gene and strongly suggests its lateral transfer from epsilon-proteobacteria. The prokaryotic origin of this enzyme might make it a promising target for therapeutics directed against Cryptosporidium.

Animals↗
PubMed2004

Inherited disorders of NF-kappaB-mediated immunity in man.

The transcription factors of the NF-kappaB family play an important role in immunity to infection in animal models. Three human primary immunodeficiencies associated with impaired NF-kappaB signaling were recently described. X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the gene encoding NEMO/IKKgamma, the regulatory subunit of the IkappaB-kinase (IKK) complex. Autosomal dominant EDA-ID (AD-EDA-ID) is caused by a hypermorphic mutation in the gene encoding the inhibitory protein IkappaBalpha. Autosomal recessive immunodeficiency without EDA is caused by mutations in the gene encoding IRAK-4, a kinase acting upstream from the IKK complex in the TIR signaling pathway. The description of the infectious phenotypes associated with these genetic defects has initiated the forward genetic dissection of NF-kappaB-mediated immunity in man.

Animals↗
PubMed2004

Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function.

Traditional gene knock-out approaches using homologous recombination in embryonic stem cells are routinely used to provide functional information about genes involved in reproduction. In the present study, we examined a novel approach using N-ethyl-N-nitrosourea (ENU) together with a balancer chromosome mating strategy to identify new loci with functional roles in male fertility. Our genetic strategy is a forward-genetic approach; thus, our phenotypic investigation begins with the discovery of an abnormal phenotype without previous knowledge of the mutant locus. We isolated eight recessive mutations on chromosome 11 that resulted in male or female infertility from a screen of 184 founder pedigrees from ENU-treated males. After testing the six male infertile and two female infertile mutations for their ability to complement, we found that three independent recessive male infertile mutations failed to complement each other. The male infertility was associated with reduced epididymal sperm count, a block in late-spermatid differentiation, and increased apoptosis. Furthermore, the three male infertile mutants had severe defects in epididymal sperm morphology associated with incorrect microtubule assembly. Electron microscopy revealed unique defects in sperm head and tail morphology for each of the three alleles. One allele had an abnormal manchette assembly of the sperm head. The other two alleles had different abnormalities in the 9+2 patterning of the microtubules in the sperm tail axoneme, with one containing only five of the microtubule doublets and the other containing an extra doublet. The isolation of this allelic series identifies a new locus on mouse chromosome 11 that is required for spermiogenesis and male fertility.

Animals↗
PubMed2004

Conserved function of medaka pink-eyed dilution in melanin synthesis and its divergent transcriptional regulation in gonads among vertebrates.

Medaka is emerging as a model organism for the study of vertebrate development and genetics, and its effectiveness in forward genetics should prove equal to that of zebrafish. Here, we identify by positional cloning a gene responsible for the medaka i-3 albino mutant. i-3 larvae have weakly tyrosinase-positive cells but lack strongly positive and dendritic cells, suggesting loss of fully differentiated melanophores. The region surrounding the i-3 locus is syntenic to human 19p13, but a BAC clone covering the i-3 locus contained orthologs located at 15q11-13, including OCA2 (P). Medaka P consists of 842 amino acids and shares approximately 65% identity with mammalian P proteins. The i-3 mutation is a four-base deletion in exon 13, which causes a frameshift and truncation of the protein. We detected medaka P transcripts in melanin-producing eyeballs and (putative) skin melanophores on embryos and an alternatively spliced form in the non-melanin-producing ovary or oocytes. The mouse p is similarly expressed in gonads, but not alternatively spliced. This is the first isolation of nonmammalian P, the functional mechanism of action of which has not yet been elucidated, even in mammals. Further investigation of the functions of P proteins and the regulation of their expression will provide new insight into body color determination and gene evolution.

Amino Acid Sequence↗
PubMed2002

It takes guts: the Drosophila hindgut as a model system for organogenesis.

The Drosophila hindgut is fruitful territory for investigation of events common to many types of organogenesis. The development of the Drosophila hindgut provides, in microcosm, a genetic model system for studying processes such as establishment (patterning) of an epithelial primordium, its internalization by gastrulation, development of left--right asymmetric looping, patterning in both the anteroposterior and dorsoventral axes, innervation, investment of an epithelium with mesoderm, reciprocal epitheliomesenchymal interactions, cell shape change, and cell rearrangement. We review the genetic control of these processes during development of the Drosophila hindgut, and compare these to related processes in other bilaterians, particularly vertebrates. We propose that caudal/Cdx, brachyenteron/Brachyury, fork head/HNF-3, and wingless/Wnt constitute a conserved "cassette" of genes expressed in the blastopore and later in the gut, involved in posterior patterning, cell rearrangement, and gut maintenance. Elongation of the internalized Drosophila hindgut primordium is similar to elongation of the archenteron and also of the entire embryonic axis (both during and after gastrulation), as well as of various tubules (e.g., nephric ducts, Malpighian tubules), as it is driven by cell rearrangement. The genes drumstick, bowl, and lines (which encode putative transcriptional regulators) are required for this cell rearrangement, as well as for spatially localized gene expression required to establish the three morphologically distinct subregions of the hindgut. Expression of signaling molecules regulated by drumstick, bowl, and lines, in particular of the JAK/STAT activator Unpaired at the hindgut anterior, may play a role in controlling hindgut cell rearrangement. Other cell signaling molecules expressed in the hindgut epithelium are required to establish its normal size (Dpp and Hh), and to establish and maintain the hindgut visceral mesoderm (Wg and Hh). Both maternal gene activity and zygotic gene activity are required for asymmetric left--right looping of the hindgut. Some of the same genes (caudal and brachyenteron) required for embryonic hindgut development also act during pupation to construct a new hindgut from imaginal cells. Application of the plethora of genetic techniques available in Drosophila, including forward genetic screens, should identify additional genes controlling hindgut development and thus shed light on a variety of common morphogenetic processes.

Animals↗
PubMed1998

Forward and reverse genetic approaches to synaptogenesis.

Genetic approaches at the neuromuscular synapse are leading investigation into the mechanisms of synaptogenesis. The marriage of classical (forward) and reverse genetic techniques allows the isolation and analysis of novel proteins involved in synaptic maturation and the functional in vivo characterization of previously identified synaptic proteins. Of particular interest are recent advances using mouse reverse genetics and Drosophila forward genetics.

Animals↗
PubMed2006

Search for solutions: animal models of disease.

Disease researchers use animal models to study potential etiologies, pathophysiologies, and treatments. Responsive models must be predictable, emulating human conditions, and produce results that can be extrapolated and transposed. These models are often better than in vitro study or computer models, but still have limitations. Animal models can be difficult to maintain and are usually quite costly. Numerous methods are used to develop animal models including chemical exposure, genetic "knockout or knock in," or forward genetic modeling. Recently, more accurate animal models of Alzheimer's disease, osteoporosis, cancer, and mental illness have been developed. The strengths and limitations of each are discussed.

Animals↗