共查询到20条相似文献,搜索用时 15 毫秒
1.
Amy C. Groth 《Journal of biological education》2019,53(3):265-273
Undergraduate laboratory exercises addressing aspects of cancer biology such as increased cell proliferation, gain-of-function signaling mutations and tumour formation often rely on tissue culture or even small mammal models. Many departments have limited or no access to these tools, and even well-equipped departments face logistical problems when incorporating these models into laboratory classes. I have developed a laboratory exercise using the microscopic worm, C. elegans, to demonstrate the effects of Notch receptor mutations on cell proliferation. Notch, which is activated by juxtacrine signaling, is mutated in many human cancers. In this exercise, students compare the germline phenotypes of worms that have a loss-of-function Notch mutation (no cells in the germline) or a gain-of-function Notch mutation (over-proliferation resulting in a germline tumour). Students also genotype the worms and perform sequence analysis to determine the effects of the mutations on the protein sequence. This laboratory exercise demonstrates oncogenic proliferation, correlates genotype to phenotype, exposes students to model organisms and introduces sequence databases and analysis. In addition to cancer biology courses, this exercise could be incorporated in courses with a focus on genetics, cell biology or developmental biology. 相似文献
2.
Open-ended, inquiry-based multiweek laboratory exercises are the key elements to increasing students' understanding and retention of the major biological concepts. Including original research into undergraduate teaching laboratories has also been shown to motivate students and improve their learning. Here, we present a series of original laboratory exercises on fine mapping novel maize mutations producing interesting phenotypes. In this 4-week lab series, students get involved in the whole process of identifying novel genes controlling specific phenotypes, from phenotype characterization and choosing appropriate molecular markers to calculating the genetic distance between the mutation and the marker and finding possible candidate genes using a complete genome sequence. We chose to use maize mutant lines produced by TILLING project. These lines have been partially mapped to a chromosomal bin by a high-throughput bulk segregant analysis; however, the exact map positions for these mutations have never been determined. Mapping these novel maize mutations provides students with the opportunity to conduct original research as a part of their classroom experience and to contribute to the field of maize genetics. The laboratory series was well received by the students, and the assessment results demonstrated an improvement of student learning of gene mapping, molecular marker analysis, and positional cloning concepts. 相似文献
3.
This laboratory exercise uses a simple preparation and a straightforward protocol to illustrate many of the basic principles of vascular biology covered in an introductory physiology course. The design of this laboratory allows students to actively participate in an exercise demonstrating the regulation of arterial tone by endothelial and extrinsic factors. In addition, this hands-on laboratory allows students to gather data using well-known basic biomedical research techniques. Specifically, students are introduced to an isolated organ-chamber technique that is widely used to study cellular mechanisms of many tissues including vascular smooth muscle contraction and dilation. On the basis of student evaluations, participation in the experiments and interpreting data reinforce lecture materials on smooth muscle and endothelial cell function and illustrate mechanisms regulating vascular tone. Students come away with a greater understanding of vascular biology, a deeper appreciation of integrative physiology, and an understanding of the process of conducting tissue-chamber experiments. 相似文献
4.
Random mutagenesis screens for recessive phenotypes require three generations of breeding, using either a backcross (BC) or
intercross (IC) strategy. Hence, they are more costly and technically demanding than those for dominant phenotypes. Maximizing
the return from these screens requires maximizing the number of mutations that are bred to homozyosity in the G3 generation. Using a probabilistic approach, we compare different designs of screens for recessive phenotypes and the impact
each one has on the number of mutations that can be effectively screened. We address the issue of BC versus IC strategies
and consider genome-wide, region-specific screens and suppressor screens. We find that optimally designed BC and IC screens
allow the screening of, on average, similar numbers of mutations but that interpedigree variation is more pronounced when
the IC strategy is employed. By conducting a retrospective analysis of published mutagenesis screens, we show that, depending
on the strategy, a threefold difference in the numbers of mutations screened per animal used could be expected. This method
allows researchers to contrast, for a range of experimental designs, the cost per mutation screened and to maximize the number
of mutations that one can expect to screen in a given experiment. 相似文献
5.
Steven O. Moldin Mary E. Farmer Hemin R. Chin James F. Battey Jr. 《Mammalian genome》2001,12(8):575-581
In the post-genomic era, the laboratory mouse will excel as a premier mammalian system to study normal and disordered biological
processes, in part because of low cost, but largely because of the rich opportunities that exist for exploiting genetic tools
and technologies in the mouse to systematically determine mammalian gene function. Many robust models of human disease may
therefore be developed, and these in turn will provide critical clues to understanding gene function. The full potential of
the mouse for understanding many of the neural and behavioral phenotypes of relevance to neuroscientists has yet to be realized.
With the full anatomy of the mouse genome at hand, researchers for the first time will be able to move beyond traditional
gene-by-gene approaches and take a global view of gene expression patterns crucial for neurobiological processes. In response
to an action plan for mouse genomics developed on the basis of recommendations from the scientific community, seven institutes
of the National Institutes of Health (NIH) initiated in 1999 a mouse genetics research program that specifically focused on
neurobiology and complex behavior. The specific goals of these neuroscience initiatives are to develop high-throughput phenotyping
assays and to initiate genome-wide mutagenesis projects to identify hundreds of mutant strains with heritable abnormalities
of high relevance to neuroscientists. Assays and mutants generated in these efforts will be made widely available to the scientific
community, and such resources will provide neuroscientists unprecedented opportunities to elucidate the molecular mechanisms
of neural function and complex behavior. Such research tools ultimately will permit the manipulation and analysis of the mouse
genome, as a means of gaining insight into the genetic bases of the mammalian nervous system and its complex disorders.
Received: 10 April 2001 / Accepted: 23 April 2001 相似文献
6.
Lu Y Savage LJ Ajjawi I Imre KM Yoder DW Benning C Dellapenna D Ohlrogge JB Osteryoung KW Weber AP Wilkerson CG Last RL 《Plant physiology》2008,146(4):1482-1500
In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected. 相似文献
7.
O'Rourke SM Yochem J Connolly AA Price MH Carter L Lowry JB Turnbull DW Kamps-Hughes N Stiffler N Miller MR Johnson EA Bowerman B 《Genetics》2011,189(3):767-778
Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems. 相似文献
8.
J. Arvid Ågren Brandon E. Campitelli Jill Wheeler 《Journal of biological education》2017,51(3):228-236
Recent years have seen a dramatic increase in our understanding of the social behaviour of microbes. Here, we take advantage of these developments to present an undergraduate laboratory exercise that uses the cooperative flocculating behaviour of yeast (Saccharomyces sp.) to introduce the concept of inclusive fitness and teach the genetics of cooperation. Students generate their own data using co-cultures of various yeast strains and perform statistical analyses to test whether kin selection or greenbeard effects determine the cooperative flocculating behaviour. The lab has run successfully for two consecutive years in a second year course with some 1, 200 students per year at the University of Toronto, Canada. We discuss the benefits of using microbes to teach social evolution, describe the set-up and learning outcomes of the laboratory exercise, and then outline possible extension and variants of the lab. In addition to providing students with the opportunity to use a model organism to study social behaviour, students are also taught common laboratory skills, such as replica plating and sterile techniques. Ultimately, while the genetics of cooperation has traditionally been taught through computer simulations and evolutionary games, this exercise demonstrates a way to experimentally introduce the topic. 相似文献
9.
Vallen E 《Cell biology education》2002,1(4):173-192
The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a chimeric protein. The chimera contains a signal sequence fused to histidinol dehydrogenase. A strain with a defect in the translocation of secretory proteins into the endoplasmic reticulum (ER) accumulates sufficient histidinol dehydrogenase in the cytoplasm to grow on media lacking histidine. In contrast, yeast proficient for secretion, or yeast with secretion defects later in the pathway, are unable to grow on media lacking histidine. Student analysis of the experimental yeast transformants and appropriate controls allows investigation into the effects of conditional defects in the secretory pathway on both cell viability and protein localization. The exercise is usually performed in a manner that allows students to execute a number of techniques common in molecular biology laboratories, including plasmid minipreps, restriction digestions, and Southern blots. Student understanding and enjoyment of the exercise was assessed by laboratory reports, oral and written examinations, and questionnaires. After completion of these experiments, students can describe the utility of protein fusions, the roles of mutant analysis in cell biology, and the steps taken by proteins transiting the secretory pathway. 相似文献
10.
The guppy, Poecilia reticulata, is one of the most famous tropical ornamental fish in the world. There are many varieties and breeds which have been bred by aqua lists for many years. The guppy has frequently been used as model organism for experiments in fish genetics because of its short life cycle, ease of breeding and reproductive capacity. The laboratory strains which were created from local (Japanese) strains and varieties were used as an experimental genetic model for aquaculture. These laboratory strains were maintained as closed colonies for five to 20 years in our laboratory. One of the roles of model experiments in fish genetics to perform experiments which take a long time in which are commercially important, such as genetic drift, as the effect of inbreeding during the growth and maintenance of population. In this review, we mainly considered the results obtained in our laboratory relating to genetic drift, and the effects of inbreeding, heterosis and inheritance of quantitative traits. 相似文献
11.
Hentges KE Nakamura H Furuta Y Yu Y Thompson DM O'Brien W Bradley A Justice MJ 《Gene expression patterns : GEP》2006,6(6):653-665
Mutagenesis screens are a valuable method to identify genes that are required for normal development. Previous mouse mutagenesis screens for lethal mutations were targeted at specific time points or for developmental processes. Here we present the results of lethal mutant isolation from two mutagenesis screens that use balancer chromosomes. One screen was localized to mouse chromosome 4, between the STS markers D4Mit281 and D4Mit51. The second screen covered the region between Trp53 and Wnt3 on mouse chromosome 11. These screens identified all lethal mutations in the balancer regions, without bias towards any phenotype or stage of death. We have isolated 19 lethal lines on mouse chromosome 4, and 59 lethal lines on chromosome 11, many of which are distinct from previous mutants that map to these regions of the genome. We have characterized the mutant lines to determine the time of death, and performed a pair-wise complementation cross to determine if the mutations are allelic. Our data suggest that the majority of mouse lethal mutations die during mid-gestation, after uterine implantation, with a variety of defects in gastrulation, heart, neural tube, vascular, or placental development. This initial group of mutants provides a functional annotation of mouse chromosomes 4 and 11, and indicates that many novel developmental phenotypes can be quickly isolated in defined genomic intervals through balancer chromosome mutagenesis screens. 相似文献
12.
C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens. 相似文献
13.
Background
Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map.Results
Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb.Conclusion
The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens. 相似文献14.
A decade after the human genome sequence, most vertebrate gene functions remain poorly understood, limiting benefits to human health from rapidly advancing genomic technologies. Systematic in vivo functional analysis is ideally suited to the experimentally accessible Xenopus embryo, which combines embryological accessibility with a broad range of transgenic, biochemical, and gain-of-function assays. The diploid X. tropicalis adds loss-of-function genetics and enhanced genomics to this repertoire. In the last decade, diverse phenotypes have been recovered from genetic screens, mutations have been cloned, and reverse genetics in the form of TILLING and targeted gene editing have been established. Simple haploid genetics and gynogenesis and the very large number of embryos produced streamline screening and mapping. Improved genomic resources and the revolution in high-throughput sequencing are transforming mutation cloning and reverse genetic approaches. The combination of loss-of-function mutant backgrounds with the diverse array of conventional Xenopus assays offers a uniquely flexible platform for analysis of gene function in vertebrate development. 相似文献
15.
Bianca V Gapp Tomasz Konopka Thomas Penz Vineet Dalal Tilmann Bürckstümmer Christoph Bock Sebastian MB Nijman 《Molecular systems biology》2016,12(8)
Reverse genetic screens have driven gene annotation and target discovery in model organisms. However, many disease‐relevant genotypes and phenotypes cannot be studied in lower organisms. It is therefore essential to overcome technical hurdles associated with large‐scale reverse genetics in human cells. Here, we establish a reverse genetic approach based on highly robust and sensitive multiplexed RNA sequencing of mutant human cells. We conduct 10 parallel screens using a collection of engineered haploid isogenic cell lines with knockouts covering tyrosine kinases and identify known and unexpected effects on signaling pathways. Our study provides proof of concept for a scalable approach to link genotype to phenotype in human cells, which has broad applications. In particular, it clears the way for systematic phenotyping of still poorly characterized human genes and for systematic study of uncharacterized genomic features associated with human disease. 相似文献
16.
《Fungal Biology Reviews》2018,32(4):249-264
Fungal model species have contributed to many aspects of modern biology, from biochemistry and cell biology to molecular genetics. Nevertheless, only a few genes associated with morphological development in fungi have been functionally characterized in terms of their genetic or molecular interactions. Evolutionary developmental biology in fungi faces challenges from a lack of fossil records and unresolved species phylogeny, to homoplasy associated with simple morphology. Traditionally, reductive approaches use genetic screens to reveal phenotypes from a large number of mutants; the efficiency of these approaches relies on profound prior knowledge of the genetics and biology of the designated development trait—knowledge which is often not available for even well-studied fungal model species. Reductive approaches become less efficient for the study of developmental traits that are regulated quantitatively by more than one gene via networks. Recent advances in genome-wide analysis performed in representative multicellular fungal models and non-models have greatly improved upon the traditional reductive approaches in fungal evo-devo research by providing clues for focused knockout strategies. In particular, genome-wide gene expression data across developmental processes of interest in multiple species can expedite the advancement of integrative synthetic and systems biology strategies to reveal regulatory networks underlying fungal development. 相似文献
17.
David C. Hess Chad L. Myers Curtis Huttenhower Matthew A. Hibbs Alicia P. Hayes Jadine Paw John J. Clore Rosa M. Mendoza Bryan San Luis Corey Nislow Guri Giaever Michael Costanzo Olga G. Troyanskaya Amy A. Caudy 《PLoS genetics》2009,5(3)
Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods. 相似文献
18.
19.
David Wilson‐Sánchez Silvia Rubio‐Díaz Rafael Muñoz‐Viana José Manuel Pérez‐Pérez Sara Jover‐Gil María Rosa Ponce José Luis Micol 《The Plant journal : for cell and molecular biology》2014,79(5):878-891
The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T‐DNA is responsible for the phenotype. To quickly map non‐annotated T‐DNA insertions, we developed a reliable, cost‐effective and easy method based on whole‐genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf ( http://genetics.umh.es/phenoleaf ) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal–distal cell cycle‐driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ. 相似文献
20.
Imparting a basic understanding of evolutionary principles to students in an active, engaging fashion can be troublesome because
the logistics involved in designing experiments where students pose their own questions and use the data to test alternative
hypotheses often outstrip time and financial constraints. In recent years, educators have begun publishing exercises that
teach evolution using innovative, in-class experiments. This article adds to this growing forum by describing a classroom
exercise that introduces the concept of evolution by natural selection in a hypothesis-driven, experimental fashion, using
a deck of cards. Our standard exercise is suitable for upper-level high school and introductory biology students at the college
level. In this paper, we discuss the exercise in detail and give several examples that illustrate how our games provide accessible
bridges to the primary literature. Finally, we discuss how extensions of our basic exercise can be used to effectively teach
advanced evolutionary concepts. 相似文献