CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery Cas9 and synthesis guide RNA (gRNA) remain as steps can limit overall ease use. Here we describe methods rapid gRNA protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into variety cells through liposome-mediated transfection or electroporation. Using these methods, report nuclease-mediated indel rates up to 94% in Jurkat T...

While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step seamless genome editing. Under conditions described here, we achieved up 56% targeted integration efficiency with six-nucleotide HEK293 cells. In induced pluripotent stem (iPSCs), editing rates 45% by co-delivering Cas9 RNP and donor DNA. addition, use short double stranded DNA...

Differentiation of oligodendrocyte progenitor cells requires activation the Src family kinase Fyn. The signals that are upstream and downstream Fyn in oligodendrocytes remain essentially unknown. Here we show extracellular matrix engagement regulates morphology activates Infection primary cultures with recombinant adenovirus revealed expression or its target p190RhoGAP induced process extension. This phenotypic change was not observed when kinase-inactive GAP-defective p190 mutants were...

Fatty acylation of Src family kinases is essential for localization the modified proteins to plasma membrane and rafts. It has been suggested that presence saturated fatty acyl chains on conducive their insertion into liquid ordered lipid domains present in The ability unsaturated dietary acids be attached not investigated. Here we demonstrate heterogeneous occurs nature acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight...

To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells to establish optimal conditions transfection. Using a systematic approach, we screened 60 transfection reagents using six commonly-used cell lines identified novel reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, genome modification efficiencies in CRISPRMAX-transfected were 40 or 15 % higher than those 3000 RNAiMAX-transfected lines,...

GAP-43 (neuromodulin) is a protein kinase C substrate that abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the N terminus has been implicated directing membrane binding. Here, we use mass spectrometry to examine stoichiometry of molecular identity fatty acid(s) attached vivo. expressed either PC12 or COS-1 cells was acetylated N-terminal methionine. Approximately 35% peptides were also modified by palmitate and/or stearate on Cys...

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTADDITION / CORRECTIONThis article has been corrected. View the notice.Vitamins and Especially Flavonoids in Common Beverages Are Powerful Vitro Antioxidants Which Enrich Lower Density Lipoproteins Increase Their Oxidative Resistance after ex Vivo Spiking Human PlasmaJoe A. Vinson, Jinhee Jang, Jihong Yang, Yousef Dabbagh, Xiquan Liang, Mamdouh Serry, John Proch, Songhuai CaiView Author Information Department of Chemistry, The University...

p190RhoGAP and Rho are key regulators of oligodendrocyte differentiation. The gene encoding is located at 19q13.3 the human chromosome, a locus that deleted in 50%–80% oligodendrogliomas. Here we provide evidence may suppress gliomagenesis by inducing differentiated glial phenotype. Using cell culture model autocrine loop PDGF stimulation, show reduced activity via overexpression or kinase inhibition induced cellular process extension, block proliferation, expression neural precursor marker...

The Gateway recombination system is characterized by its ability to transfer DNA sequences back and forth between an intermediate clone (the entry clone) a variety of destination vectors. However, number applications do not need exploit the advantages offered clone. Here we report reaction conditions for cloning fragments into vectors in single step reaction, thus reducing cost overall time needed obtain expression from three days one.

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma targeting. Most contain an Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate palmitate after removal methionine. Previous studies kinase fatty have relied on radiolabeling cells radioactive acids. Although this method useful verifying a given acid attached to protein, it does not reveal whether other acids or modifying groups are the protein. Here we use...

More than 50% of all major drug targets are membrane proteins, and their role in cell-cell interaction signal transduction is a vital concern. By culturing normal malignant breast cancer cells with light or heavy isotopes amino acids (SILAC), followed by cell fractionation, 1D gel separation crude analysis the digests using nanoelectrospray LC-MS/MS, we have quantified 1600 gene products that group into 997 protein families approximately 830 membrane-associated proteins; 100 unknown,...

Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with pathogenesis of human chronic myelogenous leukemia (CML). It difficult to characterize response stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically proteins light heavy tyrosine, we...

Protein phosphorylation regulates many aspects of cellular function, including cell proliferation, migration, and signal transduction. An efficient strategy to isolate phosphopeptides from a pool unphosphorylated peptides is essential global characterization using mass spectrometry. We describe an approach employing isotope tagging reagents for relative absolute quantification (iTRAQ) labeling compare quantitatively commercial prototypal immobilized metal affinity chelate (IMAC) oxide...

Dok-1 is an adaptor protein that a substrate for Bcr-Abl and other tyrosine kinases. The presence of pleckstrin homology phosphotyrosine binding domains as well multiple phosphorylation sites suggests involved in protein-protein and/or protein-lipid interactions. Here we show stimulation Mo7 hematopoietic cells with c-Kit ligand (KL) induces phosphatidylinositol (PI) 3-kinase-dependent membrane recruitment Dok-1. Addition the K-Ras membrane-targeting motif to generated constitutively...

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids cell culture technology to identify and quantify differentially released into the extracellular media by pair of normal breast-cancer lines. Approximately 380 non-redundant were quantified serum-free media. Of assigned proteins, 62% are classified secreted protein databases an additional 25% designated literature. A number growth factors...

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR cascade. The attenuation of early signaling events these compounds has been recently attributed a selective displacement key proteins from membrane lipid rafts. In this study, we demonstrate that GC displace acyl-bound adaptor linker for cells and phosphoprotein associated glycosphingolipid-enriched microdomains rafts murine hybridomas, possibly inhibiting their...

The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic by rat mitochondria was studied to determine whether the presence a trans double bond in place cis or no affects β-oxidation. Rat from liver heart effectively degraded coenzyme A derivatives all three fatty acids. However, with elaidoyl-CoA as substrate, major metabolite accumulated mitochondrial matrix. This isolated identified 5-trans-tetradecenoyl-CoA. In contrast, little none corresponding metabolites...

We have developed an efficient method for the simultaneous introduction of up to three mutations in a plasmid DNA via homologous recombination. The strategy is compatible with variety mutations, including degenerate codons plasmids different sizes. In contrast other methodologies, this approach employs same set reagents both single- and multi-site mutagenesis assays, minimizes required protocol steps, exhibits remarkably high efficiencies.

A number of attempts have been made to simplify the synthesis whole chromosomes generate artificial microorganisms. However, sheer size average bacterial genome makes task virtually impracticable. major limitation is maximum assembly DNA imposed by current available technologies. We propose fragment chromosome into autonomous replicating units so that (i) each episome becomes small enough be assembled in its entirety within an host and (ii) complete set should able a viable cell. In this...

Polyphenols and particularly flavonoids are well known in vitro antioxidants. Their consumption foods has been shown to decrease the risk of heart disease epidemiological studies. We examined two commonly consumed nonalcoholic juices (grape juice orange juice) for their ability act as plasma antioxidants, enrich lower-density lipoproteins after spiking, protect these from oxidation supplementation healthy subjects. found that grape juice, but not possesses all antioxidant properties is an...

The enzymes of mitochondrial beta-oxidation are thought to be organized in at least two functional complexes, a membrane-bound, long-chain-specific system and matrix consisting soluble with preferences for medium-chain short-chain substrates. This hypothesis is supported by the observation that inactivation long-chain 3-ketoacyl-CoA thiolase 4-bromotiglic acid (4-bromo-2-methylbut-2-enoic acid) causes complete inhibition palmitate even though thiolase, which acts on 3-ketopalmitoyl-CoA,...