Molecular Biology

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    Protocols in Current Issue
    Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
    Authors:  Jingen Zhu, Pan Tao, Marthandan Mahalingam and Venigalla B. Rao, date: 04/05/2020, view: 349, Q&A: 0
    [Abstract] A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we ...
    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 589, Q&A: 0
    [Abstract] The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...
    A Novel Dual Lentiviral CRISPR-based Transcriptional Activation System for Gene Expression Regulation in Neurons
    Authors:  Katherine E. Savell, Faraz A. Sultan and Jeremy J. Day, date: 09/05/2019, view: 1745, Q&A: 0
    [Abstract] Robust and efficient gene expression control enables the study of a gene’s function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we describe a protocol to generate high-titer lentiviruses with neuron-optimized CRISPR-activation ...
    Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain
    Authors:  Hiroshi Yamaguchi and Luis de Lecea, date: 08/20/2019, view: 1379, Q&A: 0
    [Abstract] Recently developed gene editing technologies based on engineered CRISPR/Cas9 systems enables researchers to disrupt genes in a cell type-specific manner in the adult mouse brain. Using these technologies, we recently showed that the dopamine beta-hydroxylase gene in Locus Coeruleus (LC) norepinephrine neurons plays a vital role in the maintenance ...
    pNP Transgenic RNAi System Manual in Drosophila
    Authors:  Fang Wang, Huan-Huan Qiao, Rong-Gang Xu, Jin Sun, Ruibao Zhu, Decai Mao and Jian-Quan Ni , date: 02/05/2019, view: 2306, Q&A: 0
    [Abstract] Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority ...
    flySAM Transgenic CRISPRa System Manual
    Authors:  Yu Jia, Da Shen, Xia Wang, Jin Sun, Ping Peng, Rong-Gang Xu, Bowen Xu and Jian-Quan Ni , date: 01/20/2019, view: 3048, Q&A: 0
    [Abstract] Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed ...
    Generation of Stable Expression Mammalian Cell Lines Using Lentivirus
    Authors:  Neha Tandon, Kaushik N. Thakkar, Edward L LaGory, Yu Liu and Amato J Giaccia, date: 11/05/2018, view: 6807, Q&A: 0
    [Abstract] Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3rd generation lentivirus is very robust and it typically takes about 1-2 ...
    Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap
    Authors:  Jonas Mechtersheimer, Stefan Reber and Marc-David Ruepp, date: 10/20/2018, view: 3299, Q&A: 0
    [Abstract] This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any ...
    Hypoxia Reporter Element Assay
    Authors:  Daelynn R Buelow and Sharyn D. D Baker, date: 08/05/2018, view: 2942, Q&A: 0
    [Abstract] Hypoxia is a condition in which there is a decrease in oxygen supply to the cellular environment. Changes to cellular oxygen levels can lead to transcriptional changes of oxygen-regulated genes. Reporter assays are used to study gene expression alteration and modifications in response to environmental changes. Dual-reporter assays allow the ...
    Overrepresentation Analyses of Differentially Expressed Genes in the Smut Fungus Ustilago bromivora during Saprophytic and in planta Growth
    Authors:  Angelika Czedik-Eysenberg, Franziska Rabe, Heinz Ekker, Carmen Czepe and Armin Djamei, date: 08/05/2017, view: 5166, Q&A: 0
    [Abstract] We have established the Ustilago bromivoraBrachypodium spp. interaction as a new model pathosystem for biotrophic fungal plant infections of the head smut type (Rabe et al., 2016). In this protocol, the methodology used for comparing gene expression between saprophytic and in planta growth of the fungus is ...



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