Protocols in Current Issue
    A Functionally Robust Phenotypic Screen that Identifies Drug Resistance-associated Genes Using 3D Cell Culture
    Authors:  Sun-Young Lee and Mina J. Bissell, date: 11/20/2018, view: 1365, Q&A: 0
    [Abstract] Drug resistance is a major obstacle in cancer treatment: A case in point is the failure of cancer patients to respond to tyrosine kinase inhibitors (TKI) of EGFR, a receptor that is highly expressed in many cancers. Identification of the targets and delineation of mechanisms of drug resistance remain major challenges. Traditional pharmacological ...
    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: 1584, 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 ...
    Quantitative ChIP-seq by Adding Spike-in from Another Species
    Authors:  Kongyan Niu, Rui Liu and Nan Liu, date: 08/20/2018, view: 2264, Q&A: 0
    [Abstract] Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of ...
    Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells
    Author:  Simona Giunta, date: 04/05/2018, view: 2614, Q&A: 0
    [Abstract] Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers (Mitelman et al., 1997; Padilla-Nash et al., 2001). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies ...
    Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 5394, Q&A: 1
    [Abstract] In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...
    Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells
    Authors:  Nicolas Brosseau, Emil Andreev and Dindial Ramotar, date: 09/20/2017, view: 2887, Q&A: 0
    [Abstract] Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.
    Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
    Authors:  Fuqiang Chen, Xiao Ding, Yongmei Feng, Timothy Seebeck, Yanfang Jiang and Gregory D Davis, date: 08/05/2017, view: 4306, Q&A: 0
    [Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous ...
    DNA Fiber Assay upon Treatment with Ultraviolet Radiations
    Authors:  Alfano Luigi, Antonio Giordano and Francesca Pentimalli, date: 06/05/2017, view: 6864, Q&A: 0
    [Abstract] Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well ...
    Formation of Minimised Hairpin Template-transcribing Dumbbell Vectors for Small RNA Expression
    Authors:  Xiaoou Jiang and Volker Patzel, date: 06/05/2017, view: 4111, Q&A: 0
    [Abstract] A major barrier for using non-viral vectors for gene therapy is the short duration of transgene expression in postmitotic tissues. Previous studies showed transgene expression from conventional plasmid fell to sub-therapeutic level shortly after delivery even though the vector DNA was retained, suggesting transcription was silenced in vivo ...
    Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
    Authors:  Hélène Tourrière, Julie Saksouk, Armelle Lengronne and Philippe Pasero , date: 06/05/2017, view: 5948, Q&A: 0
    [Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical ...

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