Cold Spring Harbor protocols最新文献_第7页

Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli. 生成用于噬菌体展示的抗体库：制备电活性大肠杆菌

Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108599

Haiyong Peng, Christoph Rader

{"title":"Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent E. coli.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108599","DOIUrl":"https://doi.org/10.1101/pdb.prot108599","url":null,"abstract":"The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 109-1011 independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F+ or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459801","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

Phage Display Selection of Antibody Libraries: Panning Procedures. 抗体库的噬菌体展示筛选：筛选程序。

Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108602

Haiyong Peng, Christoph Rader

引用次数: 0

Generation of Antibody Libraries for Phage Display: Human Fab Format. 生成用于噬菌体展示的抗体库：人类 Fab 格式。

Cold Spring Harbor protocols Pub Date : 2024-10-15 DOI: 10.1101/pdb.prot108597

Haiyong Peng, Christoph Rader

{"title":"Generation of Antibody Libraries for Phage Display: Human Fab Format.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108597","DOIUrl":"https://doi.org/10.1101/pdb.prot108597","url":null,"abstract":"Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142459799","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

Classical Conditioning of Adult Drosophila. 成年果蝇的经典条件反射

Cold Spring Harbor protocols Pub Date : 2024-10-10 DOI: 10.1101/pdb.prot108566

Zeynep Okray, Pedro F Jacob, John-Paul Moszynski, Clifford B Talbot, Scott Waddell

{"title":"Classical Conditioning of Adult Drosophila.","authors":"Zeynep Okray, Pedro F Jacob, John-Paul Moszynski, Clifford B Talbot, Scott Waddell","doi":"10.1101/pdb.prot108566","DOIUrl":"https://doi.org/10.1101/pdb.prot108566","url":null,"abstract":"Olfactory classical conditioning paradigms have been extensively used since the early 1970s to apply genetic approaches to the study of memory in Drosophila. Over the intervening years, advances in genetics have largely changed the focus of studies from the role of single genes in memory to investigation of memory-relevant neuronal circuits. However, the original behavioral paradigms have remained largely unaltered, besides investigators making a few useful tweaks to the training and testing apparatus and modifications to the operating procedures. In this protocol, we provide the reader with a detailed description of the manufacture and assembly of a typical T-maze apparatus, where populations of adult flies can be trained and their odor memory tested later, by giving them a binary choice between the two trained odors. We describe how variations of the training apparatus permit both aversive (odor-shock) and appetitive (odor-sugar) memories to be studied. In addition, we describe a recent modification of the apparatus and protocol that permits study of multisensory (color and odor) aversive and appetitive learning. Control assays for sensory acuity and locomotion are also included.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399629","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

Twists to Classical Conditioning of Adult Drosophila. 成年果蝇经典条件反射的转折

Cold Spring Harbor protocols Pub Date : 2024-10-10 DOI: 10.1101/pdb.top108567

Zeynep Okray, Scott Waddell

{"title":"Twists to Classical Conditioning of Adult Drosophila.","authors":"Zeynep Okray, Scott Waddell","doi":"10.1101/pdb.top108567","DOIUrl":"https://doi.org/10.1101/pdb.top108567","url":null,"abstract":"Memory has been extensively studied in Drosophila since the early 1970s. Straightforward aversive and appetitive conditioning paradigms train populations of flies to associate the pairing of one of two odors with either punishment or reward. After training, the flies show either preferential avoidance or approach behavior, to the appropriate odor, when given a choice between the two odors in a simple T-maze apparatus. These basic experimental approaches have proven useful in understanding the genetic, molecular, cellular, and neuronal network bases of various valence-specific memories in the fly brain. In addition, numerous modifications to these assays have permitted the study of a broad range of memory-related phenomena. Labile short-term avoidance and approach memories can be readily distinguished from more stable \"consolidated\" long-term memory equivalents. Prior or subsequent experience of the training cues, and manipulations of the flies' condition, have revealed how parallel competing memories and incompatible states can temporarily interfere with memory retrieval, providing insight into mechanisms of forgetting. Recent studies have also modified the training and testing apparatus to allow simultaneous presentation of odors and colors, providing insight into mechanisms of multisensory learning.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142399630","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

Mosquito Egg Development and Eggshell Formation. 蚊子卵的发育和卵壳的形成

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.top107669

Jun Isoe, Michael A Riehle, Roger L Miesfeld

{"title":"Mosquito Egg Development and Eggshell Formation.","authors":"Jun Isoe, Michael A Riehle, Roger L Miesfeld","doi":"10.1101/pdb.top107669","DOIUrl":"10.1101/pdb.top107669","url":null,"abstract":"Anautogenous female mosquitoes, which ingest a blood meal from warm-blooded vertebrates to produce eggs, have become a valuable model organism for investigating signaling pathways and physiological processes that occur during egg development. Different molecular pathways tightly regulate the initiation of egg development and are governed by a balance among different insect hormones. Gravid (mature egg-carrying) females deposit fully developed eggs at the end of each gonotrophic cycle, which is defined as the time interval between the ingestion of a blood meal to oviposition. An intact eggshell protects the oocyte and embryo inside from external factors such as desiccation, physical damage, etc., and the various eggshell proteins are spatially and temporary deposited during oogenesis. Additionally, follicle resorption (oosorption) during blood meal-induced mosquito ovarian follicle development is an adapted physiological process that optimizes reproductive fitness. Mosquito oocytes grow and mature synchronously throughout oogenesis; however, during the later stages of oogenesis, some oocytes may undergo oosorption if sufficient nutrients are unavailable. This introduction highlights how mosquito egg development can be used to investigate follicular resorption and identify proteins involved in eggshell formation in Aedes aegypti mosquitoes.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107669"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139402194","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

Mosquito Blood Meal Analysis. 蚊子血餐分析。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.top107706

Lawrence E Reeves, Nathan D Burkett-Cadena

{"title":"Mosquito Blood Meal Analysis.","authors":"Lawrence E Reeves, Nathan D Burkett-Cadena","doi":"10.1101/pdb.top107706","DOIUrl":"10.1101/pdb.top107706","url":null,"abstract":"The host associations of mosquitoes vary by species, with some species being relative generalists, whereas others specialize, to varying extents, on a particular subset of the available host community. These host associations are driving factors in transmission dynamics of mosquito-vectored pathogens. For this reason, characterizing the host associations of mosquito species is critical for understanding the epidemiology of mosquito-vectored pathogens. Diverse methods have been used to associate mosquito species with their hosts. These typically include collecting mosquitoes that bite a restrained host (bait) or collecting wild blood-engorged mosquitoes and matching their blood meal to reference samples (blood meal analysis). Blood meal analysis refers to a collection of molecular techniques for determining the taxonomic identity of the source of a mosquito blood meal using cytological, serological, or DNA-based characteristics of the blood meal. Blood meal analyses that are based on DNA markers have advantages over cytological and serological methods and are effective for determining species-level identities of hosts from a broad range of potential host taxa. Here, we discuss effective techniques for analyzing blood meals.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107706"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9830047","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

Generating and Validating Transgenic Mosquitoes with Transposon-Mediated Transgenesis. 转座子介导转基因蚊子的产生和验证。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108194

Vanessa Bottino-Rojas, Anthony A James

引用次数: 0

Mosquito Transposon-Mediated Transgenesis. 蚊子转座子介导的转基因。

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.top107687

Vanessa Bottino-Rojas, Anthony A James

{"title":"Mosquito Transposon-Mediated Transgenesis.","authors":"Vanessa Bottino-Rojas, Anthony A James","doi":"10.1101/pdb.top107687","DOIUrl":"10.1101/pdb.top107687","url":null,"abstract":"Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as \"enhancer traps\" to identify endogenous genes with specific expression characteristics.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.top107687"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11025883/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41193785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis. 通过液相色谱-串联质谱（LC-MS/MS）蛋白质组分析鉴定埃及伊蚊卵壳蛋白质

Cold Spring Harbor protocols Pub Date : 2024-10-01 DOI: 10.1101/pdb.prot108227

Jun Isoe, Krishna Parsawar, Cynthia L David, Michael A Riehle, Roger L Miesfeld

{"title":"Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.","authors":"Jun Isoe, Krishna Parsawar, Cynthia L David, Michael A Riehle, Roger L Miesfeld","doi":"10.1101/pdb.prot108227","DOIUrl":"10.1101/pdb.prot108227","url":null,"abstract":"The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":"pdb.prot108227"},"PeriodicalIF":0.0,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139402193","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