Tag: HEK293T

Unexpected finding in mucus clearance system may inform future pulmonary disease treatment

Generation and validation of MUC5B/AC-KO cultures. (A) Overview of the MUC5B/MUC5AC gene-targeting approach via lentivirus-mediated delivery of sgRNA and CRISPR-Cas9. Transduced and sorted cells were differentiated at air-liquid interface (ALI). Once fully differentiated, the mucus gel was collected by apically washing cultures. HEK293T, human embryonic kidney–293T; FACS, fluorescence-activated cell sorting;…

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Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

Discovery of 11 new Cas12a candidates from a human gut microbiome database To discover new members of the Cas12a protein family, we built a pipeline that searches and sorts out new candidates from the National Center for Biotechnological Information (NCBI) human metagenome database. Using this search pipeline (Fig. 1a), we initially…

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Versatile and efficient genome editing with Neisseria cinerea Cas9

PAM identification of NcCas9 by PAM-DOSE The type II-C Cas9 orthologue NcCas9 is of small size (1082 aa) and is closely related to conventional Neisseria meningitidis Cas9 (NmeCas9)11 (Fig. 1a). The NcCas9 is 94% identical to Nme1Cas9, and the divergences lie mainly in the C-terminal PAM-interacting domain (PID) (Fig. 1a and S1)….

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Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors

Robust C-to-T conversion by Cas12a variant-mediated CBEs for non-canonical PAMs We initially attempted to screen for optimal cytosine deaminases that confer powerful C-to-T editing for Cas12a-mediated CBEs. Seven dCas12a-CBEs fused with individual cytosine deaminases1,2,16,20,21,22,24,25,26 were compared in HEK293T cells and porcine embryos at sites with TTTV PAM sequences. The dCas12a-A3A…

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Evasion of cGAS and TRIM5 defines pandemic HIV

Cells and reagents HEK293T and U87 cells were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Labtech) and 100 U ml−1 penicillin plus 100 μg ml−1 streptomycin (Pen/Strep; Gibco). THP-1-IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously62. THP-1…

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Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine

Animals and immunizations The study protocol and all veterinarian procedures were approved by the Bioqual IACUC per a memorandum of understanding with the Duke IACUC, and were performed based on standard operating procedures. Macaques studied were housed and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited…

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Proscillaridin A induces mitochondrial damage and autophagy in pancreatic cancer and reduces the stability of SMAD4 in Panc-1 cells – Hou

Introduction Pancreatic cancer (PC) is one of the most aggressive and lethal cancers (1). Previous studies have shown that aging, environmental and behavioral changes are more closely related to the dramatic increase in the incidence of PC worldwide than genetic factors (2–5). A position paper published by the European Federation…

Continue Reading Proscillaridin A induces mitochondrial damage and autophagy in pancreatic cancer and reduces the stability of SMAD4 in Panc-1 cells – Hou

Transcription-independent regulation of STING activation and innate immune responses by IRF8 in monocytes

Reagents, antibodies, viruses and cells LMW-Poly(I:C), LPS, 2′3′-cGAMP and DMXAA (InvivoGen); hydroxyurea, camptothecin and mitomycin C (MCE); GM-CSF, Flt3L (peproTech); lipofectamine 2000 (Invitrogen); polybrene (Millipore); RNAiso Plus (Takara); HT-DNA (Sigma); SYBR (BIO-RAD); dual-specific luciferase assay kit (Promega); ELISA kit for murine Ifn-β (PBL); ELISA kits for murine IP-10 (Biolegend) were…

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WGS Facilitates Gene Editing System Upgrade

Researchers at the Korean Institute of Life Sciences and Technology engineered an efficient, miniaturized CRISPR-Cas gene-editing system that may be more easily packed into vectors for clinical applications. Their system employs the Cas variant Cas12f1 with a guide RNA (gRNA) remodeled to mitigate off-target effects, a design that could potentially…

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Team develops method to increase gene editing efficiency while minimizing DNA deletion sizes

Targeting E. coli DNA pol I to DSBs increased the ratio of 1-bp deletions versus >1-bp deletions and TIS versus non-TIS. (A) Counteracting DNA resection (e.g. MRE11) by pol I fused to Cas9. The expected result is suppression of the MMEJ and SSA DNA repair pathways, which require DNA resection. (B)…

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IJMS | Free Full-Text | Systematic Discovery of FBXW7-Binding Phosphodegrons Highlights Mitogen-Activated Protein Kinases as Important Regulators of Intracellular Protein Levels

Int. J. Mol. Sci. 2022, 23(6), 3320; doi.org/10.3390/ijms23063320 (registering DOI) Int. J. Mol. Sci. 2022, 23(6), 3320; doi.org/10.3390/ijms23063320 (registering DOI) Received: 23 February 2022 / Revised: 16 March 2022 / Accepted: 17 March 2022 / Published: 19 March 2022 Round 1 Reviewer 1 Report This manuscript by Singh and co-workers defines a…

Continue Reading IJMS | Free Full-Text | Systematic Discovery of FBXW7-Binding Phosphodegrons Highlights Mitogen-Activated Protein Kinases as Important Regulators of Intracellular Protein Levels

Requirement of Xk and Vps13a for the P2X7-mediated phospholipid scrambling and cell lysis in mouse T cells

Significance The extracellular concentration of adenosine triphosphate (ATP) reaches several hundred micromoles in the inflamed tissues or tumor environment. A high concentration of ATP activates P2X7, a purinergic receptor, and induces the formation of a nonselective cation channel, accompanied by reversible phosphatidylserine (PtdSer) exposure, leading to cell lysis. Here, we…

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GJA5 Over-expression Lysate Product

Expression cell line: Human HEK293T cell line Epitope tag: C-terminal Myc/DDK* Detection Antibodies: Anti-DDK Antibody Application: Western Blot experiments. Left-Control; Right -Over-expression Lysate Description: Antigen standard for gap junction protein, alpha 5, 40kDa (GJA5), transcript variant A (transient overexpression lysate) Components: 1 vial of 100 ug gene specific transient over-expression…

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A cell-based phenotypic library selection and screening approach for the de novo discovery of novel functional chimeric antigen receptors

Cell lines and culture Unless stated otherwise, all tumor target cell lines were sourced from the ATCC via LGC Standards. Human endogenous mesothelin (MSLN)-positive target cell lines H-226 (lung carcinoma, MSLN+, ATCC® CRL-5826™) and AsPC-1 (ATCC CRL-1682), and MSLN-negative Raji control cells (ATCC CCL-86) were cultured in RPMI-1640 Glutamax (Life Technologies,…

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Lead exposure induces dysregulation of constitutive heterochromatin hallmarks in live cells

This article was originally published here Curr Res Toxicol. 2021 Dec 11;3:100061. doi: 10.1016/j.crtox.2021.12.001. eCollection 2022. ABSTRACT Lead (Pb) is a heavy metal contaminant commonly found in air, soil, and drinking water due to legacy uses. Excretion of ingested Pb can result in extensive kidney damages due to elevated oxidative…

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Twin Prime Editing Promises More Precise DNA Changes Without Double-Strand Breaks

NEW YORK – Researchers at the Broad Institute have developed a new prime editing method that allows for more precise replacement or excision of DNA sequences at endogenous human genomic sites, without the need for double-strand DNA breaks (DSBs). In a paper published on Thursday in Nature Biotechnology, researchers led…

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Selection of DNA Aptamers Recognizing EpCAM-Positive Prostate Cancer b

Introduction Prostate cancer (PCa) is one of the most common genitourinary system malignant tumor in men worldwide. In Asian countries, many patients with PCa are often diagnosed at an advanced stage maybe mostly because of a large population base with relatively backward economic development and imperfect cancer screening system of…

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MYO10 drives genomic instability and inflammation in cancer

INTRODUCTION Genomic instability often refers to the existence of a variety of DNA alterations, ranging from single nucleotide changes (such as base substitution, deletion, and insertion) to chromosomal rearrangements (e.g., gain or loss of a segment or the whole chromosome) (1). Loss of genome stability can lead to early onset…

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Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity

Innate immunity is the first line of defense against microbial infection. In response to RNA virus infection, mitochondrial antiviral signaling protein (MAVS) plays an important role in innate antiviral immune responses. Arginine methylation is a Post-translational Modification (PTM) in histone and non-histone proteins that can affect numerous cellular activities. Previous…

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High-purity production and precise editing of DNA base editing ribonucleoproteins

Abstract Ribonucleoprotein (RNP) complex–mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector–mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in…

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