Subjects -> BIOLOGY (Total: 3174 journals)
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    - ENTOMOLOGY (67 journals)
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    - ORNITHOLOGY (26 journals)
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    - ZOOLOGY (117 journals)

BIOLOGY (1491 journals)            First | 1 2 3 4 5 6 7 8 | Last

Showing 201 - 400 of 1720 Journals sorted alphabetically
Biological Research     Open Access   (Followers: 1)
Biological Rhythm Research     Hybrid Journal  
Biological Theory     Hybrid Journal   (Followers: 3)
Biological Trace Element Research     Hybrid Journal  
Biologicals     Full-text available via subscription   (Followers: 7)
Biologics: Targets & Therapy     Open Access   (Followers: 1)
Biologie Aujourd'hui     Full-text available via subscription  
Biologie in Unserer Zeit (Biuz)     Hybrid Journal   (Followers: 29)
Biologija     Open Access  
Biology     Open Access   (Followers: 3)
Biology and Philosophy     Hybrid Journal   (Followers: 18)
Biology Bulletin     Hybrid Journal   (Followers: 1)
Biology Bulletin Reviews     Hybrid Journal  
Biology Direct     Open Access   (Followers: 9)
Biology Letters     Full-text available via subscription   (Followers: 45)
Biology Methods and Protocols     Open Access  
Biology of Sex Differences     Open Access   (Followers: 1)
Biology of the Cell     Full-text available via subscription   (Followers: 9)
Biology Open     Open Access  
Biology, Medicine, & Natural Product Chemistry     Open Access   (Followers: 2)
Bioma : Jurnal Ilmiah Biologi     Open Access  
Biomacromolecules     Hybrid Journal   (Followers: 23)
Biomarker Insights     Open Access   (Followers: 2)
Biomarkers     Hybrid Journal   (Followers: 4)
Biomass and Bioenergy     Partially Free   (Followers: 8)
Biomaterials     Hybrid Journal   (Followers: 55)
Biomaterials Advances     Full-text available via subscription   (Followers: 24)
Biomath     Open Access  
Biomatter     Open Access  
Biomechanics and Modeling in Mechanobiology     Hybrid Journal   (Followers: 8)
Biomedical Chromatography     Hybrid Journal   (Followers: 6)
Biomedical Engineering     Hybrid Journal   (Followers: 11)
Biomedical Engineering and Computational Biology     Open Access   (Followers: 11)
BioMedical Engineering OnLine     Open Access   (Followers: 4)
Biomedical Engineering: Applications, Basis and Communications     Hybrid Journal   (Followers: 4)
Biomedical Journal     Open Access   (Followers: 5)
Biomedical Science and Engineering     Open Access   (Followers: 5)
Biomedical Signal Processing and Control     Hybrid Journal   (Followers: 9)
BioMetals     Hybrid Journal   (Followers: 1)
Biometrical Letters     Open Access  
Biometrics     Hybrid Journal   (Followers: 50)
Biometrika     Hybrid Journal   (Followers: 21)
Biomimetic Intelligence and Robotics     Open Access  
Biomolecular NMR Assignments     Hybrid Journal   (Followers: 3)
Biomolecules     Open Access   (Followers: 1)
BioNanoScience     Partially Free   (Followers: 3)
Bionature     Open Access   (Followers: 1)
Biopreservation and Biobanking     Hybrid Journal   (Followers: 2)
Bioprocess and Biosystems Engineering     Hybrid Journal   (Followers: 8)
Bioresource Technology     Partially Free   (Followers: 9)
BioRisk     Open Access   (Followers: 2)
Biosaintifika : Journal of Biology & Biology Education     Open Access  
BioScience     Hybrid Journal   (Followers: 26)
Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science     Hybrid Journal   (Followers: 3)
Biosemiotics     Hybrid Journal   (Followers: 1)
Biosensors     Open Access   (Followers: 3)
Biosensors and Bioelectronics     Hybrid Journal   (Followers: 26)
Biosensors and Bioelectronics : X     Open Access   (Followers: 2)
Bioseparation     Hybrid Journal   (Followers: 1)
Biosfer : Jurnal Biologi dan Pendidikan Biologi     Open Access  
Biosfer : Jurnal Tadris Biologi     Open Access  
BioSocieties     Hybrid Journal   (Followers: 3)
Biospecies     Open Access  
BIOspektrum     Hybrid Journal   (Followers: 4)
Biostatistics     Hybrid Journal   (Followers: 18)
Biosystematics and Ecology     Open Access   (Followers: 5)
Biosystems     Hybrid Journal   (Followers: 3)
Biosystems Diversity     Open Access  
Biota Amazônia     Open Access  
Biota Neotropica     Open Access  
Biotechnology Advances     Hybrid Journal   (Followers: 33)
Biotropia : The Southeast Asian Journal of Tropical Biology     Open Access  
Biotropica     Hybrid Journal   (Followers: 19)
Birth Defects Research     Hybrid Journal  
BJHM Open Research     Full-text available via subscription   (Followers: 5)
BMC Bioinformatics     Open Access   (Followers: 119)
BMC Biology     Open Access   (Followers: 51)
BMC Developmental Biology     Open Access   (Followers: 13)
BMC Evolutionary Biology     Open Access   (Followers: 59)
BMC Genomics     Open Access   (Followers: 69)
BMC Molecular and Cell Biology     Open Access   (Followers: 40)
BMC Proceedings     Full-text available via subscription   (Followers: 2)
BMC Research Notes     Open Access   (Followers: 3)
BMC Structural Biology     Open Access   (Followers: 8)
BMC Systems Biology     Open Access   (Followers: 16)
Boletín Científico : Centro de Museos. Museo de Historia Natural     Open Access  
Boletín del Centro de Investigaciones Biológicas     Open Access  
Boletín Micológico     Open Access  
Bone Reports     Open Access  
Bonorowo Wetlands     Open Access  
Borneo Journal of Resource Science and Technology     Open Access  
Bothalia : African Biodiversity & Conservation     Open Access  
Brain Science Advances     Open Access  
Brazilian Journal of Biological Sciences     Open Access  
Breastfeeding Medicine     Hybrid Journal   (Followers: 20)
Briefings in Bioinformatics     Hybrid Journal   (Followers: 43)
Briefings in Functional Genomics     Hybrid Journal   (Followers: 3)
British Poultry Abstracts     Hybrid Journal   (Followers: 3)
Brittonia     Hybrid Journal  
Bulletin de la Société Royale des Sciences de Liège     Open Access  
Bulletin of Experimental Biology and Medicine     Hybrid Journal  
Bulletin of Mathematical Biology     Hybrid Journal   (Followers: 9)
Bulletin of the Ecological Society of America     Open Access   (Followers: 4)
Bulletin of the Lebedev Physics Institute     Hybrid Journal  
Butlletí de la Institució Catalana d'Història Natural     Open Access  
CABI Agriculture and Bioscience     Open Access   (Followers: 2)
Caldasia     Open Access  
Cameroon Journal of Experimental Biology     Open Access  
Canadian Journal of Bioethics     Open Access  
Canadian Journal of Plant Pathology     Hybrid Journal   (Followers: 3)
Çanakkale Onsekiz Mart University Journal of Marine Sciences and Fisheries     Open Access  
Cancer Biology & Therapy     Open Access   (Followers: 11)
Cancer Cell International     Open Access   (Followers: 7)
Carbon Capture Science & Technology     Open Access  
Carbon Management     Hybrid Journal   (Followers: 5)
Carbon Resources Conversion     Open Access   (Followers: 2)
Caryologia : International Journal of Cytology, Cytosystematics and Cytogenetics     Partially Free  
Caucasiana     Open Access  
Cell     Full-text available via subscription   (Followers: 1152)
Cell Adhesion & Migration     Open Access   (Followers: 9)
Cell and Tissue Banking     Hybrid Journal   (Followers: 1)
Cell and Tissue Biology     Hybrid Journal   (Followers: 4)
Cell and Tissue Research     Hybrid Journal   (Followers: 5)
Cell Biochemistry and Function     Hybrid Journal   (Followers: 7)
Cell Biology and Development     Open Access   (Followers: 3)
Cell Biology and Toxicology     Hybrid Journal   (Followers: 10)
Cell Biology Education     Free   (Followers: 4)
Cell Biology International     Hybrid Journal   (Followers: 4)
Cell Biology International Reports     Hybrid Journal   (Followers: 2)
Cell Calcium     Hybrid Journal   (Followers: 2)
Cell Communication & Adhesion     Hybrid Journal   (Followers: 2)
Cell Cycle     Full-text available via subscription   (Followers: 5)
Cell Death and Differentiation     Hybrid Journal   (Followers: 7)
Cell Discovery     Open Access   (Followers: 2)
Cell Division     Open Access   (Followers: 1)
Cell Genomics     Full-text available via subscription   (Followers: 3)
Cell Metabolism     Full-text available via subscription   (Followers: 58)
Cell Proliferation     Open Access  
Cell Reports     Open Access   (Followers: 62)
Cell Reports Medicine     Open Access   (Followers: 4)
Cell Reports Methods     Open Access   (Followers: 1)
Cell Research     Hybrid Journal   (Followers: 11)
Cell Stress and Chaperones     Hybrid Journal   (Followers: 1)
Cell Surface     Open Access  
Cell Systems     Hybrid Journal   (Followers: 9)
Cells     Open Access   (Followers: 2)
Cells & Development     Hybrid Journal   (Followers: 3)
Cells Tissues Organs     Full-text available via subscription   (Followers: 1)
Cellular Immunology     Hybrid Journal   (Followers: 29)
Cellular Logistics     Full-text available via subscription  
Cellular Microbiology     Hybrid Journal   (Followers: 12)
Cellular Oncology     Hybrid Journal   (Followers: 3)
Cellular Reprogramming     Hybrid Journal  
Cellular Signalling     Hybrid Journal   (Followers: 10)
Ceylon Journal of Science     Open Access  
Channels     Open Access   (Followers: 1)
Check List : The Journal of Biodiversity Data     Open Access   (Followers: 2)
Chem     Hybrid Journal   (Followers: 1)
ChemBioEng Reviews     Full-text available via subscription   (Followers: 3)
Chemosensory Perception     Hybrid Journal  
Chirality     Hybrid Journal  
Chromosoma     Hybrid Journal  
Chromosome Research     Hybrid Journal   (Followers: 2)
Ciencia     Open Access  
Ciencia Amazónica (Iquitos)     Open Access  
Ciência ET Praxis     Open Access  
CienciaUAT     Open Access  
Cladistics     Hybrid Journal   (Followers: 7)
Climate Change Ecology     Open Access   (Followers: 18)
Clinical Dysmorphology     Hybrid Journal  
Clinical Phytoscience     Open Access  
Clinical Proteomics     Open Access   (Followers: 3)
Clinical Spectroscopy     Open Access   (Followers: 1)
Coevolution     Open Access  
Cogent Biology     Open Access  
Cognitive Neurodynamics     Hybrid Journal   (Followers: 2)
Cold Spring Harbor Perspectives in Biology     Full-text available via subscription   (Followers: 4)
Cold Spring Harbor Protocols     Full-text available via subscription   (Followers: 5)
Communication in Biomathematical Sciences     Open Access   (Followers: 2)
Communications Biology     Open Access  
Communications in Applied Sciences     Open Access  
Communications Materials     Open Access  
Communicative & Integrative Biology     Open Access  
Community Ecology     Full-text available via subscription   (Followers: 27)
Comparative Medicine     Full-text available via subscription   (Followers: 5)
Composite Interfaces     Hybrid Journal   (Followers: 6)
Comptes Rendus : Chimie     Open Access  
Comptes Rendus Biologies     Open Access   (Followers: 1)
Computational Biology Journal     Open Access   (Followers: 6)
Computational Mathematics and Mathematical Physics     Hybrid Journal   (Followers: 5)
Computer Methods in Biomechanics and Biomedical Engineering     Hybrid Journal   (Followers: 10)
Computer Methods in Biomechanics and Biomedical Engineering : Imaging & Visualization     Hybrid Journal  
Computers in Biology and Medicine     Hybrid Journal   (Followers: 10)
Connective Tissue Research     Hybrid Journal  
Contact (CTC)     Open Access  
Contributions to Plasma Physics     Hybrid Journal   (Followers: 3)
CRISPR Journal     Hybrid Journal  
Critical Reviews in Clinical Laboratory Sciences     Hybrid Journal   (Followers: 16)
Crustaceana     Hybrid Journal   (Followers: 6)
Cryobiology     Hybrid Journal   (Followers: 3)

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Cold Spring Harbor Protocols
Journal Prestige (SJR): 1.03
Citation Impact (citeScore): 1
Number of Followers: 5  
 
  Full-text available via subscription Subscription journal
ISSN (Print) 1940-3402 - ISSN (Online) 1559-6095
Published by Cold Spring Harbor Lab Press Homepage  [8 journals]
  • Preparation of Yeast Cells for Staining

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      Authors: Rodig; S. J.
      Abstract: Staining yeast cells for the presence and location of antigens is particularly challenging. They are small, making the resolution of any antigen difficult; they have a thick cell wall that antibodies cannot penetrate and that is difficult to remove; and they grow in suspension, making handling difficult. In addition, background problems can be especially severe, particularly with polyclonal antibodies, because many antisera contain antibodies to yeast cell wall components. In this protocol, yeast cells are treated with paraformaldehyde, the cell wall is removed by enzymic digestion, and the spheroplasts are attached to poly-l-lysine-coated slides. After cell lysis, the cells are ready to be stained as per normal. Except in unusual circumstances, the detection reagent should be fluorochrome-labeled.
      Keywords: Cell Biology, general, Analysis of Protein Expression in Cultured Cells, Yeast, Immunostaining, Immunostaining, general, Immunostaining Cells, Antibodies, Second Edition
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot099648
      Issue No: Vol. 2022, No. 6 (2022)
       
  • Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with Whole
           Cells to Detect Cell-Surface Binding

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      Authors: Greenfield; E. A.
      Abstract: If the antigen of interest is a cell-surface protein, flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to these proteins. Two alternative protocols are presented here—staining in individual tubes and staining in 96-well plates.
      Keywords: Antibodies, general, Hybridomas and Myelomas, Monoclonal Antibodies, Immunology, general, Antibodies, Immunostaining, Immunostaining, general, Flow Cytometry, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot103077
      Issue No: Vol. 2022, No. 6 (2022)
       
  • Hybridoma Screening by Antibody Capture: Flow Cytometry/FACS with
           Permeabilized Cells to Detect Intracellular Binding

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      Authors: Greenfield; E. A.
      Abstract: Flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to internal cellular proteins, but the cells must be permeabilized before the hybridoma supernatants are applied. In using this technique, useful controls are positive and negative cell lines with primary and secondary antibodies as well as positive and negative cell lines with secondary antibody alone.
      Keywords: Antibodies, general, Hybridomas and Myelomas, Monoclonal Antibodies, Immunology, general, Antibodies, Immunostaining, Immunostaining, general, Flow Cytometry, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot103085
      Issue No: Vol. 2022, No. 6 (2022)
       
  • Generating Nonmosaic Mutants in Xenopus Using CRISPR-Cas in Oocytes

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      Authors: Cha; S.-W.
      Abstract: In CRISPR–Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR–Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR–Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR–Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR–Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73–104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.
      Keywords: Preparation of Macromolecules and Introduction into Cells, Genetics, general, Xenopus, Molecular Biology, general, Mutagenesis, Developmental Biology, Collections, Xenopus, DNA Delivery/Gene Transfer, DNA Delivery/Gene Transfer, general, Xenopus
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot106989
      Issue No: Vol. 2022, No. 6 (2022)
       
  • I-SceI-Mediated Transgenesis in Xenopus

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      Authors: Noble, A; Abu-Daya, A, Guille, M. J.
      Abstract: Transgenic frogs can be very efficiently generated using I-SceI meganuclease, a nuclease with an 18-bp recognition site. The desired transgene must be flanked by I-SceI sites, in either a plasmid or a polymerase chain reaction (PCR) product. After a short in vitro digestion with the meganuclease, the complete reaction is injected into fertilized eggs, where the enzyme mediates genomic integration by an unknown mechanism. Posttransgenesis development is typically normal, and up to 70% of the embryos integrate the transgene.
      Keywords: Cell Biology, general, Xenopus, Transgenic Technology, general, Xenopus Transgenics, Developmental Biology, Collections, Xenopus, DNA Delivery/Gene Transfer, DNA Delivery/Gene Transfer, general, Use of Reporter Genes, Xenopus
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot107011
      Issue No: Vol. 2022, No. 6 (2022)
       
  • Size Quantification of Blood and Sugar Meals in Aedes aegypti Mosquitoes

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      Authors: Venkataraman, K; Jove, V, Duvall, L. B.
      Abstract: Both male and female mosquitoes consume sugar-rich nectar meals required for metabolic energy, but only females consume protein-rich blood meals, which are required for egg development. The size of each meal consumed has subsequent effects on behavior and reproduction; therefore, precise quantification is an important aspect of mosquito feeding behavior studies. This protocol describes a high-throughput, end-point assay to quantify meal volumes ingested by individual mosquitoes. The addition of a fluorescent dye to the meal allows for meal size quantification. Individual mosquitoes that have been fed this meal are homogenized in 96-well plates, and the fluorescence levels are measured with a plate reader. This protocol can also be adapted to determine if alteration of meal composition affects the ingested meal volume, if mosquito strain or genotype dictates consumption, or if meals are derived from multiple sources.
      Keywords: Neuroscience, general, Behavioral Assays, Mosquitoes, Mosquitoes
      PubDate: 2022-06-24T08:50:29-07:00
      DOI: 10.1101/pdb.prot107862
      Issue No: Vol. 2022, No. 6 (2022)
       
  • Hybridoma Screening by Antibody Capture: Enzyme-Linked Detection (Indirect
           ELISA) in Polyvinyl Chloride Wells

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      Authors: Greenfield; E. A.
      Abstract: In this antibody capture assay for hybridoma screening, the antigen is immobilized on a solid substrate (the surface of the wells in a polyvinyl chloride [PVC] microtiter plate), and antibodies in the hybridoma tissue culture supernatant are incubated with the antigen. Unbound antibodies are removed by washing, and antibody–antigen complexes are detected by secondary antibody conjugated to alkaline phosphatase (AP), which catalyzes the conversion of a chromogenic substrate to a blue/green product. Alternative secondary antibodies are necessary for experiments in which immunoglobulin-fusion proteins have been used as immunogens or screening proteins.
      Keywords: Antibodies, general, Hybridomas and Myelomas, Monoclonal Antibodies, Immunology, general, Antibodies, Immunoassay, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot103044
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Hybridoma Screening by Antibody Capture: Immunohistochemistry

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      Authors: Greenfield; E. A.
      Abstract: To determine the subcellular location of an antigen, hybridoma tissue culture supernatants can be screened using immunohistochemistry. For antibodies to have access to antigens in fixed and embedded tissue sections, the paraffin must be removed and the tissue must be rehydrated and digested before immunohistochemical staining.
      Keywords: Antibodies, general, Hybridomas and Myelomas, Monoclonal Antibodies, Immunology, general, Antibodies, Immunohistochemistry, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot103119
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony
           Double-Diffusion Assays

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      Authors: Greenfield; E. A.
      Abstract: Originally, the Ouchterlony double-diffusion assays were the most common method for determining the class and subclass of a monoclonal antibody, and they still are useful, particularly when only a few assays will be performed. A sample of hybridoma tissue culture supernatant is placed in a well in a bed of agar, and class- and subclass-specific antisera are placed in other wells in a ring surrounding the test antibody. As the antibodies diffuse into the agar, they meet and multimeric immune complexes precipitate to form a visible "precipitin line."
      Keywords: Antibodies, general, Handling Antibodies, Hybridomas and Myelomas, Handling Antibodies, general, Monoclonal Antibodies, Antibodies, Immunoassay, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot103218
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Determining the Class and Subclass of Monoclonal Antibodies Using Antibody
           Capture on Antigen-Coated Plates

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      Authors: Greenfield; E. A.
      Abstract: The class or subclass of an antibody is defined by its heavy chain. There are five main classes of antibodies: M, G, A, E, and D. By definition, a monoclonal antibody should only be of a single class or subclass. Each class of antibody is associated with specific functions. The method of antibody purification will differ based on the class. In this protocol, antigen is used to capture antibodies reactive to it. Specific class and/or subclass secondary antibodies are then used to discern which class/subclass has been captured.
      Keywords: Antibodies, general, Handling Antibodies, Hybridomas and Myelomas, Handling Antibodies, general, Monoclonal Antibodies, Antibodies, Immunoassay, Generating Antibodies, Generating Antibodies, general, Antibodies, Second Edition
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot103226
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Xenopus Tadpole Craniocardiac Imaging Using Optical Coherence Tomography

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      Authors: Deniz, E; Mis, E. K, Lane, M, Khokha, M. K.
      Abstract: Optical coherence tomography (OCT) imaging can be used to visualize craniocardiac structures in the Xenopus model system. OCT is analogous to ultrasound, utilizing light instead of sound to create a gray-scale image from the echo time delay of infrared light reflected from the specimen. OCT is a high-speed, cross-sectional, label-free imaging modality, which can outline dynamic in vivo morphology at resolutions approaching histological detail. OCT imaging can acquire 2D and 3D data in real time to assess cardiac and facial structures. Additionally, during cardiac imaging, Doppler imaging can be used to assess the blood flow pattern in relation to the intracardiac structures. Importantly, OCT can reproducibly and efficiently provide comprehensive, nondestructive in vivo cardiac and facial phenotyping. Tadpoles do not require preprocessing and thus can be further raised or analyzed after brief immobilization during imaging. The rapid development of the Xenopus model combined with a rapid OCT imaging protocol allows the identification of specific gene/teratogen phenotype relationships in a short period of time. Loss- or gain-of-function experiments can be evaluated in 4–5 d, and OCT imaging only requires ~5 min per tadpole. Thus, we find this pairing an efficient workflow for screening numerous candidate genes derived from human genomic studies to in-depth mechanistic studies.
      Keywords: Imaging Development, Image Analysis, In Vivo Imaging, In Vivo Imaging, general, Xenopus, Collections, Xenopus, Xenopus
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot105676
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Chambers for Culturing and Immobilizing Xenopus Embryos and Organotypic
           Explants for Live Imaging

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      Authors: Chu, C.-W; Davidson, L. A.
      Abstract: Live imaging of Xenopus embryos and organotypic explants can be challenging because of their large size and slippery nature. This protocol covers the preparation of special chambers for immobilizing Xenopus embryos and embryonic explants for live-cell and tissue imaging. The opaque nature of Xenopus embryonic tissues enables simple bright-field imaging techniques for tracking surface movements across large regions. Such surface imaging of embryos or organotypic explants can directly reveal cell behaviors, obviating the need for complex postprocessing commonly required to extract this data from 3D confocal or light-sheet observations of more transparent embryos. Furthermore, Xenopus embryos may be filled with light-absorbing pigment granules and light-scattering yolk platelets, but these limitations are offset by the utilitarian nature of Xenopus organotypic explants that expose and stabilize large embryonic cells in a nearly native context for high-resolution live-cell imaging. Additionally, whole embryos can be stabilized for long-term bright-field and confocal microscopy. Simple explants can be prepared using a single cell type, and organotypic explants can be prepared in which multiple tissue types are dissected while retaining native tissue–tissue interactions. These preparations enable both in-toto imaging of tissue dynamics and super-resolution imaging of protein dynamics within individual cells. We present detailed protocols for these methods together with references to applications.
      Keywords: Cell Biology, general, Imaging/Microscopy, general, Imaging Development, In Vivo Imaging, In Vivo Imaging, general, Xenopus, Developmental Biology, Explant Culture, Collections, Xenopus, Xenopus
      PubDate: 2022-06-07T08:48:02-07:00
      DOI: 10.1101/pdb.prot107649
      Issue No: Vol. 2022, No. 5 (2022)
       
  • Obtaining Xenopus tropicalis Eggs

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      Authors: Lane, M; Mis, E. K, Khokha, M. K.
      Abstract: Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. For cell biological studies, egg extracts can mimic many processes in a cell-free system. For developmental biology, Xenopus embryos are a premier system, combining cut-and-paste embryology with modern gene manipulation tools. Xenopus tropicalis are particularly suited to genetic studies because of their diploid genome, as compared to the tetraploid genome of Xenopus laevis. When collecting eggs, there are differences in timing of steps, amounts of hormone administered, and handling of females between these species. In this protocol, X. tropicalis females are induced with a hormone that stimulates ovulation, and then eggs are collected. To administer the ovulation hormone and express eggs, it is necessary to be comfortable with handling frogs. Proficient handling of X. tropicalis requires practice, as they are relatively small, active, and slippery.
      Keywords: Laboratory Organisms, general, Xenopus, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-04-01T06:27:19-07:00
      DOI: 10.1101/pdb.prot106344
      Issue No: Vol. 2022, No. 4 (2022)
       
  • Obtaining Xenopus tropicalis Embryos by In Vitro Fertilization

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      Authors: Lane, M; Khokha, M. K.
      Abstract: Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. In vitro fertilization (IVF) is ideal for obtaining developmentally synchronized embryos for microinjection or when natural mating has failed to produce a fertilization. In IVF, females are induced to ovulate, and then eggs are collected by manual expression. After testes are collected from a euthanized male frog, the eggs are fertilized in vitro. The embryos are then treated with cysteine to remove the sticky protective jelly coat. Dejellied embryos are much easier to manipulate during microinjection or when sorting in a Petri dish. The jelly coat is also very difficult to penetrate with an injection needle. After microinjection, embryos are maintained in Petri dishes until desired stages are reached. Although in vitro fertilization in X. laevis and X. tropicalis is similar, critical differences in solutions, handling of testis, response of fertilized eggs directly after introduction of sperm, and developmental timing are required for successful fertilization in X. tropicalis.
      Keywords: Laboratory Organisms, general, Xenopus, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-04-01T06:27:19-07:00
      DOI: 10.1101/pdb.prot106351
      Issue No: Vol. 2022, No. 4 (2022)
       
  • Raising and Maintaining Xenopus tropicalis from Tadpole to Adult

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      Authors: Lane, M; Slocum, M, Khokha, M. K.
      Abstract: Xenopus tropicalis is a powerful model organism for cell and developmental biology research. Recently, precise gene-editing methods such as CRISPR–Cas9 have allowed facile creation of mutants. The ability to raise and maintain lines of wild-type and mutant animals through all life stages is thus critical for researchers using this model organism. The long fertile life (>8–10 yr) and relatively hardy nature of X. tropicalis makes this a straightforward process. Environmental parameters such as water temperature, pH, and conductivity often vary slightly among husbandry protocols. However, the stability of these variables is essential for rearing success. This protocol describes conditions to optimally raise and maintain X. tropicalis from embryos to adulthood.
      Keywords: Laboratory Organisms, general, Xenopus, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-04-01T06:27:19-07:00
      DOI: 10.1101/pdb.prot106369
      Issue No: Vol. 2022, No. 4 (2022)
       
  • Obtaining Xenopus tropicalis Embryos by Natural Mating

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      Authors: Lane, M; Khokha, M. K.
      Abstract: Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. Natural matings are a simple and common method to obtain embryos for injection or other experimental use or to raise to adulthood. This method does not require sacrificing a male as in vitro fertilization (IVF) does. Male and female frogs are injected with an ovulation hormone, placed together in a mating bucket, and left for 4–6 h or overnight to mate. Embryos are then collected, treated with cysteine to remove the sticky jelly coat, and used for injections and/or raised to later stages or adulthood. For embryos raised past free-swimming stages, the cysteine step can optionally be skipped, and tadpoles can be allowed to hatch naturally from the jelly coat. Although there are many similarities between natural mating protocols for Xenopus laevis and Xenopus tropicalis, there are key differences such as hormone dosage, timing of ovulation, and embryo incubation temperature. Here we provide a specific protocol for inducing natural matings in X. tropicalis.
      Keywords: Laboratory Organisms, general, Xenopus, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-04-01T06:27:19-07:00
      DOI: 10.1101/pdb.prot106609
      Issue No: Vol. 2022, No. 4 (2022)
       
  • Microinjection of Xenopus tropicalis Embryos

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      Authors: Lane, M; Mis, E. K, Khokha, M. K.
      Abstract: Microinjection is an important technique used to study development in the oocyte and early embryo. In Xenopus, substances such as DNA, mRNA, and morpholino oligonucleotides have traditionally been injected into Xenopus laevis, because of their large embryo size and the relatively long time from their fertilization to first division. In the past few decades, Xenopus tropicalis has become an important model in developmental biology; it is particularly useful in genetic studies. The advent and rapid development of CRISPR–Cas9 technology has provided an array of targeted gene manipulations for which X. tropicalis is particularly suited. The equipment and protocol for X. tropicalis microinjection is broadly transferable from X. laevis. There are important differences between the species to consider, however, including the smaller embryo size and faster embryo development time in X. tropicalis. There are a number of solutions and reagents that differ in concentration and composition as well. Here we describe a microinjection protocol specifically for studies in X. tropicalis.
      Keywords: Fluorescence, Fluorescence, general, Xenopus, Transgenic Technology, general, Xenopus Transgenics, Developmental Biology, Collections, Xenopus, DNA Delivery/Gene Transfer, DNA Delivery/Gene Transfer, general, Xenopus
      PubDate: 2022-04-01T06:27:19-07:00
      DOI: 10.1101/pdb.prot107644
      Issue No: Vol. 2022, No. 4 (2022)
       
  • Labeling of DNA Probes by Polymerase Chain Reaction

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      Authors: Green, M. R; Sambrook, J.
      Abstract: The polymerase chain reaction (PCR) can be used to produce both nonradiolabeled DNA probes and radiolabeled DNA probes with high specific activity. In this protocol, PCR is used to generate double-stranded probes. Related methods, including the generation of asymmetric probes by PCR, are also discussed.
      Keywords: Molecular Biology, general, Probes, Probes, general, Radiolabeled Probes, Non-isotopically Labeled Probes, Polymerase Chain Reaction (PCR), Polymerase Chain Reaction (PCR), general, Amplification of DNA by PCR, Molecular Cloning, Fourth Edition
      PubDate: 2022-03-01T06:27:30-08:00
      DOI: 10.1101/pdb.prot100610
      Issue No: Vol. 2022, No. 3 (2022)
       
  • CRISPR-Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in
           the F0 Generation and Beyond

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      Authors: Blitz, I. L; Nakayama, T.
      Abstract: CRISPR–Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis. Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR–Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR–Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.
      Keywords: Preparation of Macromolecules and Introduction into Cells, Genetics, general, Xenopus, Molecular Biology, general, Mutagenesis, RNA, RNA, general, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-03-01T06:27:30-08:00
      DOI: 10.1101/pdb.prot106971
      Issue No: Vol. 2022, No. 3 (2022)
       
  • Modeling Human Genetic Disorders with CRISPR Technologies in Xenopus

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      Authors: Willsey, H. R; Guille, M, Grainger, R. M.
      Abstract: Combining the power of Xenopus developmental biology with CRISPR-based technologies promises great discoveries in understanding and treating human genetic disorders. Here we provide a practical pipeline for how to go from known disease gene(s) or risk gene(s) of interest to methods for gaining functional insight into the contribution of these genes to disorder etiology in humans.
      Keywords: Genetics, general, Xenopus, Mutagenesis, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-03-01T06:27:30-08:00
      DOI: 10.1101/pdb.prot106997
      Issue No: Vol. 2022, No. 3 (2022)
       
  • Tissue-Targeted CRISPR-Cas9-Mediated Genome Editing of Multiple Homeologs
           in F0-Generation Xenopus laevis Embryos

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      Authors: Corkins, M. E; DeLay, B. D, Miller, R. K.
      Abstract: Xenopus laevis frogs are a powerful developmental model that enables studies combining classical embryology and molecular manipulation. Because of the large embryo size, ease of microinjection, and ability to target tissues through established fate maps, X. laevis has become the predominant amphibian research model. Given that their allotetraploid genome has complicated the generation of gene knockouts, strategies need to be established for efficient mutagenesis of multiple homeologs to evaluate gene function. Here we describe a protocol to use CRISPR–Cas9-mediated genome editing to target either single alleles or multiple alloalleles in F0 X. laevis embryos. A single-guide RNA (sgRNA) is designed to target a specific DNA sequence encoding a critical protein domain. To mutagenize a gene with two alloalleles, the sgRNA is designed against a sequence that is common to both homeologs. This sgRNA, along with the Cas9 protein, is microinjected into the zygote to disrupt the genomic sequences in the whole embryo or into a specific blastomere for tissue-targeted effects. Error-prone repair of CRISPR–Cas9-generated DNA double-strand breaks leads to insertions and deletions creating mosaic gene lesions within the embryos. The genomic DNA isolated from each mosaic F0 embryo is sequenced, and software is applied to assess the nature of the mutations generated and degree of mosaicism. This protocol enables the knockout of genes within the whole embryo or in specific tissues in F0 X. laevis embryos to facilitate the evaluation of resulting phenotypes.
      Keywords: Cell Biology, general, Preparation of Macromolecules and Introduction into Cells, Genetics, general, Xenopus, Molecular Biology, general, Mutagenesis, Xenopus Transgenics, Developmental Biology, Collections, Xenopus, Xenopus
      PubDate: 2022-03-01T06:27:30-08:00
      DOI: 10.1101/pdb.prot107037
      Issue No: Vol. 2022, No. 3 (2022)
       
 
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