Tag: GFP

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes…

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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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Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential…

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Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

Significance Tools that afford spatiotemporal control of gene expression are crucial for studying genes and processes in multicellular organisms. Stop cassettes consist of exogenous sequences that interrupt gene expression and flanking site-specific recombinase sites to allow for tissue-specific excision and restoration of function by expression of the cognate recombinase. We…

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Identification of a regulatory pathway inhibiting adipogenesis via RSPO2

Integration of APC scRNA-seq data reveals heterogeneity of adipocyte progenitor cells In a previous study9, we defined Lin−Sca1+CD142+ APCs as adipogenesis regulatory (Areg) cells and demonstrated that these cells are both refractory toward adipogenesis and control adipocyte formation of APCs through paracrine signaling. In contrast, Merrick et. al.4 observed that Lin−CD142+ cells…

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Molecular Basis for Retroviral Neuropathogenesis

Murine leukemia-inducing retroviruses (MuLVs) can undergo changes that allow them to cause neurological diseases in rodents, and understanding the molecular basis for these changes will give us insights into the genetic elements required for distinct disease phenotypes. We have been studying PVC-211 MuLV, a variant of the erythroleukemia-inducing Friend MuLV…

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Microtubule Plus-End Dynamics Visualization in Huntington’s Disease Model based on Human Primary Skin Fibroblasts

This protocol is dedicated to the microtubule plus-end visualization by EB3 protein transfection to study their dynamic properties in primary cell culture. The protocol was implemented on human primary skin fibroblasts obtained from Huntington’s disease patients. The Huntington’s disease, HD, is an incurable, neurodegenerative pathology caused by a mutation in…

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Switch to regmap for mmio access

[PATCH] pwm: pwm-mtk-disp: Switch to regmap for mmio access * [PATCH] pwm: pwm-mtk-disp: Switch to regmap for mmio access @ 2022-01-03 15:35 AngeloGioacchino Del Regno 0 siblings, 0 replies; only message in thread From: AngeloGioacchino Del Regno @ 2022-01-03 15:35 UTC (permalink / raw) To: thierry.reding Cc: u.kleine-koenig, lee.jones, matthias.bgg,…

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Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome

Significance Cu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme, and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress; metabolic defects; and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2•−) in a manner that…

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Mechanism for DNA invasion of adenoviral Covi

image: Incoming adenovirus particles (AdV) dock at the nuclear pore complexes of a human cell’s nucleus (NPC, dashed line). The cellular enzyme Mind bomb1 (Mib1, grey-white structures) primes them for unlocking and removes protein V (GFP-V, green dots). The uncoated viral DNA genome is then imported into the nucleus. The arrow…

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AAV ShRNA Cloning Service – CD Biospeeds

AAV ShRNA Cloning Service AAV ShRNA Cloning Service Adeno-associated virus (AAV) is a type of parvovirus. Its genome is single-stranded DNA and has the ability to infect both dividing and non-dividing cells. Adenovirus or herpes virus is usually needed to help it replicate and expand in the…

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Single-cell delineation of lineage and genetic identity in the mouse brain

STICR lentiviral library preparation and validation We synthesized a high-complexity lentivirus barcode library that encodes approximately 60–70 million distinct oligonucleotide RNA sequences (STICR barcodes). STICR barcodes comprised three distinct oligonucleotide fragments cloned sequentially into a multicloning site within the 3′ UTR of an enhanced green fluorescent protein (eGFP) transgene under…

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New biosensors allow CRISPR gene edits to be seen with the naked eye

New technology developed by the Department of Energy’s Oak Ridge National Laboratory (ORNL) allows the activity of CRISPR to be seen with the naked eye and an ultraviolet flashlight. In a paper published in ACS Synthetic Biology, scientists describe the development and function of this technology. Their process involves redirecting…

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Watching CRISPR-Cas9 at Work in Real-Time

Detecting the activity of CRISPR gene editing tools in organisms with the naked eye and an ultraviolet flashlight is now possible using technology developed at the Department of Energy’s Oak Ridge National Laboratory. Scientists demonstrated these real-time detection tools in plants and anticipate their use in animals, bacteria and fungi…

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New biosensors shine a light on CRISPR gene editing — ScienceDaily

Detecting the activity of CRISPR gene editing tools in organisms with the naked eye and an ultraviolet flashlight is now possible using technology developed at the Department of Energy’s Oak Ridge National Laboratory. Scientists demonstrated these real-time detection tools in plants and anticipate their use in animals, bacteria and fungi…

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New biosensor sheds light on CRISPR gene editing

ORNL’s biosensor system reveals the CRISPR activity of poplar plants that glow bright green under UV light compared to normal plants that appear red.Credits: Guoliang Yuan / ORNL, US Department of Energy Using technology developed at the Oak Ridge National Laboratory of the Department of Energy, it is now possible…

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New biosensors shine a light on CRISPR gene editing

ORNL’s biosensor system reveals CRISPR activity in poplar plants, which glow bright green under ultraviolet light, compared to normal plants, which appear red. Credit: Guoliang Yuan/ORNL, U.S. Dept. of Energy Detecting the activity of CRISPR gene editing tools in organisms with the naked eye and an ultraviolet flashlight is now…

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Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components

Available online 8 December 2021 doi.org/10.1016/j.nbt.2021.12.002Get rights and content Highlights • An efficient geminiviral vector was developed from Beet curly top virus with a wide host range. • The geminiviral vector delivered CRISPR/Cas components efficiently into plant cells. • A high frequency of both mutagenesis and complete HDR in plant…

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Epistasis shapes the fitness landscape of an allosteric specificity switch

Computational design Protein modeling and design was performed with Rosetta version 3.5 (2015.19.57819)35,37. Python and shell scripts for generating input from Rosetta and analyzing from Rosetta are available at: github.com/raman-lab/biosensor_design The high-resolution TtgR structure co-crystalized with tetracycline was selected as the starting point for computational design (PDB: 2UXH)28. The structure…

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Cristae-dependent quality control of the mitochondrial genome

S. cerevisiae cells distinguish between WT and mutant mtDNA. (A) Schematic illustration of the pedigree analysis. Two Δarg8 yeast strains harboring WT or Δcob::ARG8 mtDNA were mated. Zygotes were isolated, and daughter cells from up to five consecutive generations were separated and placed on free spots on the agar plate…

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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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ToolGen Files Opposition to Broad Preliminary Motion No. 3 to De-Designate Claims as Corresponding to Either Interference Count | McDonnell Boehnen Hulbert & Berghoff LLP

On May 28th, Junior Party the Broad Institute, Harvard University, and MIT (collectively, “Broad”) filed its Substantive Preliminary Motion No. 3 in CRISPR Interference No. 106,126 (where ToolGen is the Senior Party).  This motion, pursuant to 37 C.F.R. §§ 41.121(a)(1)(iii) and 41.208(a)(1) requested that the Board de-designate Broad claims in these…

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High tumor mutation burden and DNA repair gene mutations

Introduction Anaplastic lymphoma kinase (ALK)‑fusion genes represent a small but important part of oncogenic driver mutations in NSCLC, accounting for approximately 3%‑7% of all cases worldwide.1,2 Small molecule tyrosine kinase inhibitors (TKIs) are the standard therapy for ALK-rearranged NSCLC. Crizotinib, a first-generation TKI, is the most widely used targeted drug…

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Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

The mammalian genome is characterized by widespread methylation of cytosine residues. After fertilization, however, both maternal and paternal genomes undergo extensive demethylation, reaching a low point in the blastocyst (1⇓⇓–4). The embryo genome is then remethylated by the activity of de novo DNA methylation enzymes (5). Mouse embryonic stem (ES)…

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Mini CRISPR Genome Editing System Created

The common analogy for CRISPR gene editing is that it works like molecular scissors, cutting out select sections of DNA. Stanley Qi, assistant professor of bioengineering at Stanford University, likes that analogy, but he thinks it’s time to reimagine CRISPR as a Swiss Army knife. “CRISPR can be as simple as…

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Compact CasMINI CRISPR Tech Is Easier to Deliver to Cells, Could Have Broad Gene Therapy Potential

Scientists led by a team at Stanford University have developed a compact, efficient CRISPR-Cas system, called CasMINI, which is about half the size of existing CRISPR-Cas systems, and which could have broad utility for gene therapy applications as well as cell engineering. The researchers confirmed in experiments that CasMINI could,…

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Stanford researchers engineer an efficient, multi-purpose, mini CRISPR system

The common analogy for CRISPR gene editing is that it works like molecular scissors, cutting out select sections of DNA. Stanley Qi, assistant professor of bioengineering at Stanford University, likes that analogy, but he thinks it’s time to reimagine CRISPR as a Swiss Army knife. “CRISPR can be as simple…

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Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma

Significance The collection of large amounts of whole-genome sequencing data allowed for identification of mutational signatures, which are characteristic combinations of substitutions in the context of neighboring bases. The clinical significance of these mutational signatures is still largely unknown. In neuroblastoma, we showed that high levels of cytosine > adenine…

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ToolGen Files Opposition to CVC Contingent Responsive Preliminary Motion No. 1 | McDonnell Boehnen Hulbert & Berghoff LLP

On June 11th, Junior Party the University of California, Berkeley; the University of Vienna; and Emmanuelle Charpentier (collectively, “CVC”) filed its Responsive Preliminary Motion No. 1 in Interference No. 106,127 to be accorded benefit of priority to U.S. Patent Application No. 13/842,859, filed March 15, 2013, or in the alternative…

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ToolGen Files Opposition to CVC Substantive Preliminary Motion No. 3 to Add Claims in ToolGen Patent | McDonnell Boehnen Hulbert & Berghoff LLP

On May 20th, Junior Party the University of California, Berkeley; the University of Vienna; and Emmanuelle Charpentier (collectively, “CVC”) filed its Substantive Preliminary Motion No. 3 in Interference No. 106,127 (which names ToolGen as Senior Party), asking the Patent Trial and Appeal Board to add claims in ToolGen’s U.S. Patent…

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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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An epigenetic basis of inbreeding depression in maize

INTRODUCTION Charles R. Darwin documented inbreeding depression as growth disadvantages from self-fertilization compared to outcrossing in many plants (1). Prevailing hypotheses suggest that inbreeding depression results from the exposure of deleterious recessive alleles and/or loss of overdominant alleles due to increased homozygosity (2, 3) or reduced recombination frequency in some…

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Can I remove the control in differential expression analysis?

Hi there, Essentially, my experimental design is control vs treatment. Cells were sorted based on fluorescence, so there are 4 different “colors” of treated cells, i.e. red, green, green+red, and blue+green+red. I am interested in how the colors differ from one another. And, I have duplicates for all colors and…

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The LpoA activator is required to stimulate the peptidoglycan polymerase activity of its cognate cell wall synthase PBP1a

Significance Class A penicillin-binding proteins (aPBPs) assemble the bacterial cell wall and are the targets of penicillin and related β-lactam antibiotics. In gram-negative bacteria, the aPBPs require outer membrane lipoproteins to function. However, little is known about how these proteins promote the activity of their cognate synthases in cells. Here,…

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Electroporation ELECTROPORATION It consists of applying short high voltage

ELECTROPORATION It consists of applying short high voltage pulses to a mixture of host cells in suspension and recombinant vectors in solution. These pulses increase the permeability of the membranes, allowing the vectors to pass into the cell. It is a highly efficient method that can be applied to all…

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