Developmental Biology

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    Protocols in Current Issue
    A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol
    Author:  Chiara Sinigaglia, date: 09/05/2019, view: 302, Q&A: 0
    [Abstract] In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence–the hybridization step, followed by detection ...
    Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis
    Authors:  Leanna K. Pantier, Jiang Li and Catherine A. Christian, date: 09/05/2019, view: 361, Q&A: 0
    [Abstract] The estrous cycle provides a readout of reproductive health in female laboratory rodents, and estrous cycle stage can be an important physiological variable. Accurate assessment of estrous cycle stage is also important in producing timed pregnancies for developmental studies. Here, we provide a protocol for evaluation of estrous cycle stage ...
    Application of a Modified Smart-seq2 Sample Preparation Protocol for Rare Cell Full-length Single-cell mRNA Sequencing to Mouse Oocytes
    Authors:  Rebecca S. Treger, Scott D. Pope, Xiaojun Xing and Akiko Iwasaki, date: 08/20/2019, view: 281, Q&A: 0
    [Abstract] Endogenous retroviruses (ERV) are transposable retroelements that form ~10% of the murine genome and whose family members are differentially expressed throughout embryogenesis. However, precise regulation of ERV in germ cells remains unclear. To investigate ERV expression in oocytes, we adapted a single-cell mRNA-sequencing library preparation ...
    Ex vivo Drosophila Wing Imaginal Disc Culture and Furin Inhibitor Assay
    Authors:  Alex Sohr, Lijuan Du and Sougata Roy, date: 08/20/2019, view: 236, Q&A: 0
    [Abstract] Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are ...
    Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells
    Authors:  Jia Wang, Boon Tin Chua, Peng Li and Feng-Jung Chen, date: 07/20/2019, view: 588, Q&A: 0
    [Abstract] Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death–inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular ...
    CRISPR/Cas9 + AAV-mediated Intra-embryonic Gene Knocking in Mice
    [Abstract] Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos’ genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation ...
    Determining Oxidative Damage by Lipid Peroxidation Assay in Rat Serum
    Authors:  Qian Tang, Yu-Wen Su and Cory J. Xian, date: 06/20/2019, view: 771, Q&A: 0
    [Abstract] It has been well-established that malondialdehyde (MDA), which is generated during the process of lipid peroxidation, is a commonly known biomarker for oxidative stress. Therefore, the serum levels of MDA are detected by using the lipid peroxidation assay with commercially available kit to determine the induction of oxidative stress in rat models.
    Simultaneous Fluorescent Recordings of Extracellular ATP and Intracellular Calcium in Mammalian Cells
    Authors:  Nicholas Mikolajewicz and Svetlana V. Komarova, date: 05/20/2019, view: 1180, Q&A: 0
    [Abstract] Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the ...
    Quantification of the Humidity Effect on HR by Ion Leakage Assay
    Authors:  Musoki Mwimba and Xinnian Dong, date: 04/05/2019, view: 905, Q&A: 0
    [Abstract] We describe a protocol to measure the contribution of humidity on cell death during the effector-triggered immunity (ETI), the plant immune response triggered by the recognition of pathogen effectors by plant resistance genes. This protocol quantifies tissue cell death by measuring ion leakage due to loss of membrane integrity during the ...
    Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos
    Authors:  Rachna Narayanan and Andrew C. Oates, date: 03/20/2019, view: 2277, Q&A: 0
    [Abstract] In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to ...



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