JournalTOCs

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

[8 journals]

Preparation of Yeast Cells for Staining
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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
- Free pre-print version: Loading...
  Rate this result: What is this?
  
  Please help us test our new pre-print finding feature by giving the pre-print link a rating.
  A 5 star rating indicates the linked pre-print has the exact same content as the published article.
  
  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)