CSH protocols最新文献_第6页

Growth and maintenance of dictyostelium cells. dictyostelium细胞的生长和维持。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5099

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Growth and maintenance of dictyostelium cells.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5099","DOIUrl":"https://doi.org/10.1101/pdb.prot5099","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a \"social ameba\" because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. Dictyostelium also provides an excellent model system for the study of phagocytosis, the molecular basis of various human diseases, and the mechanisms of drug action. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Most Dictyostelium strains used in the laboratory can be grown either with bacteria or in axenic medium. When grown in the presence of bacteria, cells double approximately every 4 h, whereas axenically grown cells double more slowly, every 8-12 h. The cells can be grown in a standard microbiology incubator or on the laboratory bench, provided the room temperature is consistently ~22°C. This protocol describes a variety of methods for growing and maintaining Dictyostelium.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5099","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Extraction of DNA from dictyostelium. dictyostelium DNA的提取。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5105

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Extraction of DNA from dictyostelium.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5105","DOIUrl":"https://doi.org/10.1101/pdb.prot5105","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Extraction of genomic DNA is used to clone gene fragments and for analysis of mutants to determine the site of vector integration. Because Dictyostelium cells contain relatively high levels of carbohydrate and nucleases, commercially available DNA preparation kits are not very successful. The DNA isolated according to the following protocol is suitable for digestion by restriction enzymes, amplification by polymerase chain reaction (PCR), and Southern blotting.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5105","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

The Two-Spotted Cricket Gryllus bimaculatus: An Emerging Model for Developmental and Regeneration Studies. 双斑蟋蟀:一个发展和再生研究的新兴模型。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.emo110

Taro Mito, Sumihare Noji

{"title":"The Two-Spotted Cricket Gryllus bimaculatus: An Emerging Model for Developmental and Regeneration Studies.","authors":"Taro Mito, Sumihare Noji","doi":"10.1101/pdb.emo110","DOIUrl":"https://doi.org/10.1101/pdb.emo110","url":null,"abstract":"INTRODUCTIONThe two-spotted cricket Gryllus bimaculatus De Geer (Orthoptera: Gryllidae), which is one of the most abundant cricket species, inhabits the tropical and subtropical regions of Asia, Africa, and Europe. G. bimaculatus can be easily bred in the laboratory and has been widely used to study insect physiology and neurobiology. Recently, this species has become established as a model animal for studies on molecular mechanisms of development and regeneration because its mode of development is more typical of arthropods than that of Drosophila melanogaster, and the cricket is probably ancestral for this phylum. Moreover, the cricket is a hemimetabolous insect, in which nymphs possess functional legs with a remarkable capacity for regeneration after damage. Because RNA interference (RNAi) works effectively in this species, the elucidation of mechanisms of development and regeneration has been expedited through loss-of-function analyses of genes. Furthermore, because RNAi-based techniques for analyzing gene functions can be combined with assay systems in other research areas (such as behavioral analyses), G. bimaculatus is expected to become a model organism in various fields of biology. Thus, it may be possible to establish the cricket as a simple model system for exploring more complex organisms such as humans.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.emo110","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 69

Whole-mount in situ hybridization in amphimedon. 两栖动物全载原位杂交。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5096

Claire Larroux, Bryony Fahey, Maja Adamska, Gemma S Richards, Marie Gauthier, Kathryn Green, Erica Lovas, Bernard M Degnan

引用次数: 21

Multicellular development of dictyostelium. 盘基骨的多细胞发育。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5100

Pascale Gaudet, Petra Fey, Rex Chisholm

引用次数: 7

Transformation of dictyostelium with plasmid DNA by calcium phosphate precipitation. 磷酸钙沉淀法转化质粒DNA的dictyostium。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5102

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Transformation of dictyostelium with plasmid DNA by calcium phosphate precipitation.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5102","DOIUrl":"https://doi.org/10.1101/pdb.prot5102","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Calcium phosphate precipitation is a commonly used method for DNA-mediated transformation in Dictyostelium. Calcium phosphate precipitation produces high-copy-number transformants and is often used for overexpression experiments in conjunction with the G418 resistance gene, which needs to be present at high levels to produce efficient selection.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Extraction of RNA from dictyostelium. dictyostelium中RNA的提取。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5106

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Extraction of RNA from dictyostelium.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5106","DOIUrl":"https://doi.org/10.1101/pdb.prot5106","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. The extraction of RNA from Dictyostelium is relatively easy because RNA levels are very high in comparison to DNA levels (i.e., ~40 times higher). Certain commercially available kits, such as Trizol (Invitrogen) and RNeasy (QIAGEN) have been used successfully, although lysis conditions need to be adjusted. RNA samples are stable for several years at -80°C in diethylpyrocarbonate (DEPC)-treated H(2)O. For longer-term storage, the RNA pellet can be stored in 100% ethanol at -80°C. Such samples are suitable for Northern blots, reverse transcriptase-polymerase chain reaction (RT-PCR), and microarray analysis of gene expression.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Selection of dictyostelium transformants. dictyostelium变形菌的选择。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5104

Pascale Gaudet, Petra Fey, Rex Chisholm

{"title":"Selection of dictyostelium transformants.","authors":"Pascale Gaudet, Petra Fey, Rex Chisholm","doi":"10.1101/pdb.prot5104","DOIUrl":"https://doi.org/10.1101/pdb.prot5104","url":null,"abstract":"INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Two commonly used methods for DNA-mediated transformation in Dictyostelium are calcium phosphate precipitation and electroporation. Transformants can then be selected in liquid media or on bacterial plates. The latter method reduces the chances of contamination because the cells are grown in buffered agar containing live or dead bacteria, rather than in a rich broth. This method also facilitates the isolation of clones from transformations because each transformant produces a single colony on the plate instead of the pools of transformants obtained in liquid culture. For gene ablation experiments, it is important to obtain a minimum of two independent clones with the same phenotype to exclude the possibility that the phenotype is due to a nonspecific mutation.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29703013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides. 利用核酸可编程蛋白阵列(NAPPA)鉴定蛋白-蛋白相互作用。方案1:查询蛋白在NAPPA载玻片上的共表达。

CSH protocols Pub Date : 2008-12-01 DOI: 10.1101/pdb.prot5108

Andrew J Link, Joshua Labaer

{"title":"Using the Nucleic Acid Programmable Protein Array (NAPPA) for Identifying Protein-Protein Interactions. Protocol 1: Coexpression of Query Protein on NAPPA Slides.","authors":"Andrew J Link, Joshua Labaer","doi":"10.1101/pdb.prot5108","DOIUrl":"https://doi.org/10.1101/pdb.prot5108","url":null,"abstract":"INTRODUCTIONThe Nucleic Acid Programmable Protein Array (NAPPA) approach for producing protein microarrays uses cell-free extracts to transcribe and translate cDNAs encoding target proteins directly onto glass slides. Following array preparation, interactions with a protein of interest (query protein) are detected either by probing an expressed NAPPA slide with the purified query protein or by coexpressing the query protein on the NAPPA slide at the same time that the target proteins are expressed. This protocol describes the coexpression method, which involves adding the gene for the query protein to the cell-free protein expression mix. The amount of query protein that is transcribed and translated from the corresponding plasmid DNA depends on the amount of plasmid DNA used and the size of the protein of interest, among other factors. If too little query protein is expressed, there may be no detectable binding signal. Excessive amounts of protein expression may generate nonspecific background signals. Because the optimum amount of query plasmid varies with each query protein, it is essential to assess empirically the optimal amount of query protein DNA to add to a coexpression experiment.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.prot5108","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29701741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Comb jellies (ctenophora): a model for Basal metazoan evolution and development. 栉水母(栉水母):基础后生动物进化和发展的模型。

CSH protocols Pub Date : 2008-11-01 DOI: 10.1101/pdb.emo106

Kevin Pang, Mark Q Martindale

{"title":"Comb jellies (ctenophora): a model for Basal metazoan evolution and development.","authors":"Kevin Pang, Mark Q Martindale","doi":"10.1101/pdb.emo106","DOIUrl":"https://doi.org/10.1101/pdb.emo106","url":null,"abstract":"INTRODUCTIONCtenophores, or comb jellies, are a group of marine organisms whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. These gelatinous creatures are clearly distinct from cnidarian medusae (i.e., jellyfish). Key features present in the ctenophore body plan include biradial symmetry, an oral-aboral axis delimited by a mouth and an apical sensory organ, two tentacles, eight comb rows composed of interconnected cilia, and thick mesoglea. Other morphological features include definitive muscle cells, a nerve net, basal lamina, a sperm acrosome, and light-producing photocytes. Aspects of their development made them attractive to experimental embryologists as early as the 19th century. Recently, because of their role as an invasive species, studies on their role in ecology and fisheries-related fields have increased. Although the phylogenetic placement of ctenophores with respect to other animals has proven difficult, it is clear that, along with poriferans, placozoans, and cnidarians, ctenophores are one of the earliest diverging extant animal groups. It is important to determine if some of the complex features of ctenophores are examples of convergence or if they were lost in other animal branches. Because ctenophores are amenable to modern technical approaches, they could prove to be a highly useful emerging model.","PeriodicalId":10835,"journal":{"name":"CSH protocols","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2008-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1101/pdb.emo106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29702121","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 37