Cold Spring Harbor protocols最新文献_第9页

Preservation of Field-Collected Mosquito Blood Meals. 保存野外采集的蚊子血餐。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108290

Lawrence E Reeves, Nathan D Burkett-Cadena

{"title":"Preservation of Field-Collected Mosquito Blood Meals.","authors":"Lawrence E Reeves, Nathan D Burkett-Cadena","doi":"10.1101/pdb.prot108290","DOIUrl":"10.1101/pdb.prot108290","url":null,"abstract":"All PCR- and DNA-based blood meal analyses require host DNA from a mosquito blood meal to be effectively preserved between the time when the specimen is collected and the extraction of DNA. As soon as a mosquito ingests blood from a host animal, digestion of host cells and cellular components within the blood meal by enzymes in the mosquito midgut begins to degrade the host DNA templates that are the targets of polymerase chain reaction (PCR) amplification. Without effective preservation, host DNA is typically undetectable by PCR 48 h after feeding, because of digestion. Preservation methods for mosquito blood meals vary in their efficacy, and the logistics of fieldwork can limit the options for preservation of blood meals and maintenance of the integrity of host DNA. This protocol describes a method of blood meal preservation that is effective, convenient, and amenable to fieldwork in remote locations where cryopreservation at -20°C or -80°C may not be feasible. It uses a Flinders Technology Associates (FTA) card, which is a chemically treated card that lyses cells and allows nucleic acids to be preserved. This method is also expected to preserve the DNA or RNA of pathogens present within the engorged mosquito abdomen, including RNA viruses.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108290"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9830048","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

Amplification and Identification of Vertebrate Host Cytochrome c Oxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals. 从蚊子血餐中扩增和鉴定脊椎动物宿主细胞色素 c 氧化酶亚基 I (COI) DNA 条形码模板。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108292

Lawrence E Reeves, Nathan D Burkett-Cadena

{"title":"Amplification and Identification of Vertebrate Host Cytochrome c Oxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals.","authors":"Lawrence E Reeves, Nathan D Burkett-Cadena","doi":"10.1101/pdb.prot108292","DOIUrl":"10.1101/pdb.prot108292","url":null,"abstract":"Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108292"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9830046","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

Visualization of Apoptotic Ovarian Follicles during Aedes aegypti Mosquito Egg Maturation by Fluorescent Imaging Studies. 通过荧光成像研究观察埃及伊蚊卵子成熟过程中凋亡的卵泡。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108226

Jun Isoe, Roger L Miesfeld, Michael A Riehle

{"title":"Visualization of Apoptotic Ovarian Follicles during Aedes aegypti Mosquito Egg Maturation by Fluorescent Imaging Studies.","authors":"Jun Isoe, Roger L Miesfeld, Michael A Riehle","doi":"10.1101/pdb.prot108226","DOIUrl":"10.1101/pdb.prot108226","url":null,"abstract":"In insects, oocyte resorption (oosorption) or follicular atresia is one of the key physiological processes and evolutionary strategies used to optimize reproductive fitness. Mosquitoes are ideal model organisms for studying egg maturation in arthropods, as their follicle development is initiated only following the ingestion of a blood meal, followed by a carefully orchestrated series of hormonally regulated events leading to egg maturation. A cohort of approximately 100 follicles per mosquito ovary begin developing synchronously. However, a significant fraction of follicles ultimately undergo apoptosis and oosorption, especially when available resources from the blood meal are limited. Therefore, simple, rapid, and reliable techniques to accurately evaluate follicular atresia are necessary to understand mechanisms underlying follicle development in insects. This protocol describes how to detect apoptotic follicle cells within the Aedes aegypti mosquito ovaries using a commercially available fluorescent-labeled inhibitor of caspases (FLICA). Caspases are key players in animal apoptosis. In this assay, the FLICA reagent enters the intracellular compartment of follicles in dissected mosquito ovaries and covalently binds to active caspases. The bound reagent remains within the cell and its fluorescent signal can be observed by confocal microscopy. Although this method was specifically developed for visualizing apoptotic ovarian follicles during Ae. aegypti mosquito egg development, it should be applicable to other mosquito tissues that undergo caspase-mediated program cell death in a time-dependent manner.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108226"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139402195","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

Extracting DNA from Preserved Mosquito Blood Meals. 从保存的蚊子血餐中提取 DNA。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108291

Lawrence E Reeves, Nathan D Burkett-Cadena

{"title":"Extracting DNA from Preserved Mosquito Blood Meals.","authors":"Lawrence E Reeves, Nathan D Burkett-Cadena","doi":"10.1101/pdb.prot108291","DOIUrl":"10.1101/pdb.prot108291","url":null,"abstract":"Mosquito species vary in their host associations. Although some species are relative generalists, most specialize, to varying extents, on particular types of host animals. Mosquito host associations are among the most important factors that influence the transmission dynamics of mosquito-vectored pathogens, and understanding these associations can provide insight on how such pathogens move within ecosystems. Characterization of the host associations of mosquito species requires applying blood meal analysis to the largest possible sample size of mosquito blood meals. Processing large samples of mosquito blood meals can be time-consuming, especially when chain-termination sequencing is used, necessitating individual processing of each specimen. Various methods and commercially available kits and products are available for extracting DNA from mosquito blood meals. The hot sodium hydroxide and Tris (HotSHOT) method is a rapid and inexpensive method of DNA extraction that is compatible with the recovery of DNA from mosquito blood meals preserved on QIAcard Flinders Technology Associates (FTA) Classic Cards (FTA cards). FTA cards allow nucleic acids found in blood meals to be preserved easily, even in field conditions. DNA prepared using this method is suitable for polymerase chain reaction (PCR)-based blood meal analysis.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108291"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9827440","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

A Rapid Agrobacterium-Mediated Transformation Method Using Maize B104 Immature Embryos. 利用玉米 B104 未成熟胚胎的农杆菌介导快速转化法

Cold Spring Harbor protocols Pub Date : 2024-09-19 DOI: 10.1101/pdb.prot108595

Minjeong Kang, Mercy K Azanu, Keunsub Lee, Kan Wang

引用次数: 0

Recent Advances in Agrobacterium-Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants. 利用未成熟胚胎和幼苗叶轮外植体进行农杆菌介导的玉米遗传转化的最新进展》（New Advances in Agrobacterium-Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants.

Cold Spring Harbor protocols Pub Date : 2024-09-19 DOI: 10.1101/pdb.top108460

Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang

{"title":"Recent Advances in Agrobacterium-Mediated Maize Genetic Transformation Using Immature Embryos and Seedling Leaf Whorl Explants.","authors":"Mercy K Azanu, Minjeong Kang, Keunsub Lee, Kan Wang","doi":"10.1101/pdb.top108460","DOIUrl":"https://doi.org/10.1101/pdb.top108460","url":null,"abstract":"The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limitations in explant availability. Indeed, although the most commonly used method, immature embryo transformation, has been improved through optimization of tissue culture media composition and selection methods, the approach is only applicable to a limited number of public genotypes, including B104 and Hi II. Recently, genotype-flexible methods have been developed using coexpression cassettes of morphogenic transcription factors (MTFs) Baby boom (Bbm) and Wushel2 (Wus2), which have enabled the successful transformation of many previously recalcitrant maize lines. This MTF-based transformation method has also allowed for the use of alternate explants, such as seedling leaf whorl, whose production is cost-effective and requires only minimum controlled growth space. In this review, we summarize recent advances in Agrobacterium-mediated maize transformation methods that use immature embryos or seedling leaf whorls as starting material.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281502","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

Studying Drosophila Larval Behavior in Agarose Channels. 在琼脂糖通道中研究果蝇幼虫的行为

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108420

Marie R Greaney, Ellie S Heckscher

{"title":"Studying Drosophila Larval Behavior in Agarose Channels.","authors":"Marie R Greaney, Ellie S Heckscher","doi":"10.1101/pdb.prot108420","DOIUrl":"https://doi.org/10.1101/pdb.prot108420","url":null,"abstract":"Larvae of the fruit fly Drosophila melanogaster are a popular and tractable model system for studying the development and function of sensorimotor circuits, thanks to the relative numerical simplicity of their nervous system and the wealth of available genetic tools to manipulate the anatomy, activity, and function of specific cell types. Researchers studying the role of a particular gene or cell type in sensorimotor circuit activity or function may wish to observe the effects of an experimental manipulation during behavior in the intact animal. Observing these effects, which may include changes in the intracellular calcium concentration or movement of small numbers of neurons, muscles, etc., typically requires high-spatial-resolution imaging, which poses several difficulties in the freely crawling larva. Freely crawling larvae can move quickly and with changeable heading, making manual or automatic tracking challenging; additionally, they may make three-dimensional movements, such as rearing, that can degrade imaging focus. These challenges are potentially solvable using advanced imaging and algorithmic tracking setups, but cost, space, or development time may be prohibitive. This protocol describes a simple and cost-effective method for placing larvae inside agarose channels, thereby restricting larval crawling to a single dimension and enabling higher-magnification time-series imaging of fluorescently labeled structures during many cycles of locomotion. By using larvae that express fluorescent calcium indicators in cells of interest, researchers can apply this method to study the effects of experimental manipulations on neural or muscular activity during behavior in the intact animal.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281504","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

The Study of Sensorimotor Circuit Assembly in Drosophila melanogaster Embryos and Larvae. 黑腹果蝇胚胎和幼虫感觉运动回路组装研究

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.top108395

Chris C Wreden, Ellie S Heckscher

{"title":"The Study of Sensorimotor Circuit Assembly in Drosophila melanogaster Embryos and Larvae.","authors":"Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.top108395","DOIUrl":"https://doi.org/10.1101/pdb.top108395","url":null,"abstract":"In animals, movement is generated by the activity of motor circuits housed in the vertebrate spinal cord or the arthropod nerve cord. How motor circuits form is a fundamental question, with wide-ranging impacts on the fields of development, neurobiology, medicine, evolution, and beyond. Until recently, studying circuit assembly had been experimentally difficult, with a paucity of suitable models. Due to the introduction of novel neuroscience tools (calcium imaging, optogenetics, connectomics), Drosophila embryos and larvae can be used as models to study motor circuit assembly. Here, we briefly review the knowledge relevant to motor circuit assembly in Drosophila larvae. We discuss the larval body and its movements, larval neurons and circuits in the motor system, and how the generation of neural diversity starting from stem cells relates to circuit formation. The long-term goal of Drosophila research in this field is to identify developmental rules, determine when the rules apply, generate an integrated understanding of motor circuit development, and uncover molecular mechanisms driving the assembly process. Motor circuits are an ancient part of the nervous system, and so far, the developmental programs guiding motor circuit assembly appear to be largely conserved across phyla. Thus, as methods improve in other systems, findings in Drosophila will provide foundational concepts that will inspire hypotheses in those systems.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281505","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

Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System. 果蝇胚胎和幼虫中枢神经系统 RNA 的荧光原位杂交连锁反应。

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108423

Jake E Henderson, Chris C Wreden, Ellie S Heckscher

{"title":"Fluorescent In Situ Hybridization Chain Reaction for RNA in the Drosophila Embryonic and Larval Central Nervous System.","authors":"Jake E Henderson, Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.prot108423","DOIUrl":"https://doi.org/10.1101/pdb.prot108423","url":null,"abstract":"In the Drosophila nerve cord, much is known about the generation of neurons from neuronal stem cells. Over the lifetime of a neuron, the cumulative expression of genes within that neuron determines its fate. Furthermore, gene expression in mature neurons determines their functional characteristics. It is therefore useful to visualize neural gene expression, which is often done via staining with antibodies to a protein of interest. In cases where there is no antibody to a desired gene product, or when it is useful to detect RNA rather than protein products, fluorescent in situ hybridization chain reaction for RNA (HCR RNA-FISH, or HCR for this protocol) can be used to detect and quantify RNA expression. RNA molecules reside predominantly in the cell soma, so HCR can facilitate determining neuron identity because somata position within the nerve cord is stereotyped across animals. HCR provides high-amplitude, high-fidelity signals. In principle, HCR can be broken down into a detection/hybridization stage and an amplification stage. During detection/hybridization, a probe set hybridizes to multiple sequences within a target gene. In the amplification step, concatemerized fluorescent hairpins bind to the hybridized probes. This two-step process increases the specificity of the fluorescent signal and helps reduce the likelihood of background fluorescence compared to traditional in situ hybridization techniques where the hybridizing probe itself contains the fluorescent signal. Here, we describe a protocol for using HCR to study gene expression in the Drosophila embryonic and larval nerve cord. We also describe how to combine HCR with immunofluorescence staining.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281499","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

Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out. 利用多色 FLP-Out 对果蝇的单神经元进行标记

Cold Spring Harbor protocols Pub Date : 2024-09-16 DOI: 10.1101/pdb.prot108422

Zarion D Marshall, Chris C Wreden, Ellie S Heckscher

{"title":"Single-Neuron Labeling in Drosophila Using Multicolor FLP-Out.","authors":"Zarion D Marshall, Chris C Wreden, Ellie S Heckscher","doi":"10.1101/pdb.prot108422","DOIUrl":"https://doi.org/10.1101/pdb.prot108422","url":null,"abstract":"Neurons exhibit some of the most striking examples of morphological diversity of any cell type. Thus, when studying neurons, the morphology of each neuron must be considered individually. However, neurons densely populate the central nervous system (CNS), making it difficult to ascertain fine morphological features due to a lack of spatial resolution. In Drosophila, this problem can be partially resolved by using driver lines that express the yeast transcription factor GAL4 in subsets of neurons. GAL4 can activate the expression of other introduced genetic elements such as genes for fluorescent proteins or other markers under the control of the GAL4 upstream activation sequences (UAS effectors). However, even highly specific GAL4 lines often label sets of potentially morphologically heterogeneous neurons. Here, we describe a protocol for using the multicolor flip-out (MCFO) technique in Drosophila melanogaster to stochastically label individual neurons within a GAL4 expression pattern. MCFO relies on the binary GAL4/UAS expression system in Drosophila but adds additional control for how densely the neurons within a GAL4 expression pattern are labeled via user-controlled heat shock. Specifically, three discrete UAS effector elements containing the sequences for unique epitope tags (FLAG, HA, and V5) linked to a gene for nonfluorescent GFP can be independently expressed under the control of GAL4 only when a transcriptional stop sequence in the UAS promoter sequence has been removed by heat shock-induced recombination. This effectively labels multiple individual neurons with either one or a combination of epitope tags that can be spectrally resolved with immunofluorescence. The MCFO technique is ideal for researchers who want to determine morphological features of CNS neurons in wild-type or mutant backgrounds.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142281503","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