Mammalia

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    Protocols in Current Issue
    Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation
    [Abstract] Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in ...
    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 ...
    An Improved Staining Method for Low Signal LacZ Reporter Detection in Mouse Embryos
    Authors:  Xiaopeng Shen, Feng Xu, Shen Wu, Meng Li, Jingyi Zhang, Rui Liang and Yu Liu, date: 03/05/2019, view: 1334, Q&A: 0
    [Abstract] In many fields of biology, especially in the field of developmental biology, LacZ reporter staining is an approach used to monitor gene expression patterns. In the LacZ reporter system, the LacZ gene is inserted in the endogenous location of the target gene via gene knock-in or by constructing a transgenic cassette in ...
    mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells
    Authors:  Madara Ratnadiwakara and Minna-Liisa Änkö, date: 11/05/2018, view: 7520, Q&A: 0
    [Abstract] Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs ...
    RNA Immunoprecipitation Assay to Determine the Specificity of SRSF3 Binding to Nanog mRNA
    Authors:  Madara Ratnadiwakara and Minna-Liisa Änko, date: 11/05/2018, view: 2127, Q&A: 0
    [Abstract] Mammalian cells express hundreds of RNA binding proteins (RBPs) that are essential regulators of RNA metabolism. RBP activity plays a central role in the control of gene expression programs and identification of RNA-protein interactions is critical for comprehensive understanding of gene regulation in cells. In recent years, various tools and ...
    Isolation of LYVE-1+ Endothelial Cells from Mouse Embryos
    Author:  Patrick Crosswhite, date: 08/05/2018, view: 1964, Q&A: 0
    [Abstract] Lymphatic vessel endothelial hyaluronan receptor 1, or LYVE-1, is a type 1 integral membrane glycoprotein expressed by lymphatic endothelial cells (LECs). LYVE-1 is commonly used as a biological marker to visually distinguish developing lymphatic vessels from blood endothelial cells (arteries or veins). As our understanding of lymphatic biology is ...
    In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development
    Authors:  Changfu Yao, Gianni Carraro and Barry R. Stripp, date: 05/20/2018, view: 2901, Q&A: 0
    [Abstract] Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide ...
    Intra-amniotic Injection of Mouse Embryos
    Authors:  Liyuan Cui, Peng Zou, Qingshuo Meng, Lu Lu and Jiayi Zhang, date: 05/20/2018, view: 2435, Q&A: 0
    [Abstract] Recent outbreaks of infectious neuro-developmental diseases such as congenital Zika syndrome - have led to a demand for prognosis data from animal models. We developed an intra-amniotic injection mice model that allows Zika virus (ZIKV) infected mice to grow to puberty. In this system, ZIKV is injected into the amniotic fluid of pregnant mice and ...
    FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe
    Authors:  Shengli Dong and Suresh K Alahari, date: 04/20/2018, view: 4094, Q&A: 0
    [Abstract] This is a flow cytometry-based protocol to measure glucose uptake of mouse embryonic fibroblasts (MEFs) and breast cancer cells in vitro. The method is a slightly modified and updated version as previously described (Dong et al., 2017). Briefly, the target cells are incubated with the fluorescently tagged ...
    Organotypic Explants of the Embryonic Rodent Hippocampus: An Accessible System for Transgenesis
    Authors:  Archana Iyer , Lakshmi Subramanian and Shubha Tole, date: 03/20/2018, view: 2987, Q&A: 0
    [Abstract] This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can ...



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