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We narrowed to 1,064 results for: tra

Showing: 221 - 240 of 1064 results

  1. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone...requires continuous administration of Dox to repress transgene expression. In contrast, in the Tet-on system...membrane pores) transfection. These techniques are still used and efficiently transfer plasmid DNA efficiently...comes to transferring DNA into primary cells or non-dividing cells. To target these un-transfectable cells...property makes the lentivirus an attractive choice for a gene transfer vector. Lentiviral vectors Although... purify and concentrate your lentiviral particles to get them to the optimum concentration to infect your...lentiviral transfer vectors The most widely used inducible lentiviral vector system is the tetracycline (Tet...

  2. Sleeping Beauty Awakens for Genome Engineering

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    Blog Post
    ...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...

  3. Important Considerations When Using AAVs

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    Blog Post
    ...experiment! Traveling from the Original Location - Axonal Transport It has been demonstrated that certain...cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What tissue...their efficiency at transducing different brain regions. Although AAV2 can transduce many tissue types,...axoplasmic transport. These AAVs may move toward the cell bodies of neurons (retrograde transport) or towards...retrograde and anterograde transport, but they also have similar transport characteristics (velocities...strong CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...scAAV) (7). As single stranded DNA viruses, AAVs normally require second strand synthesis before gene ...

  4. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

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    Blog Post
    ... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...less than 10%. In contrast, the original paper introducing prime editing demonstrated ~20-50% efficiency...delivery strategy, cell type, and goal of the experiment can all impose different constraints and affect...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ...other strand. To guide heteroduplex resolution to favor the edit, the Liu lab turned to a strategy they...

  5. 28 Hot Plasmid Technologies from 2015

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    Blog Post
    ...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...endosomal trafficking to signaling pathways to the cell cycle. Good tools for measuring intracellular pH are...of its substrate, luciferin, which releases energy as a photon. The photon energy is transferred to the...ribosome during translation, which knocks off all mRNA binding proteins as it travels along the mRNA to...the stop codon. Thus a non-translated mRNA will appear yellow, but a translated mRNA will be red. This system...plasmids, Halstead et al demonstrated that 91% of reporter mRNAs were untranslated in the nucleus, confirming...P-bodies are translationally repressed whereas the nonsequestered pool goes on to initiate translation. These...

  6. CRISPR 101: Homology Directed Repair

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    Blog Post
    ...breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular factors such ...CRISPR/Cas-mediated Transgenesis (2018). Pubmed. The Road Less Traveled: Strategies to Enhance the Frequency...overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA...DNA repair synthesis. The ssDNA strand then displaces one strand of the homologous DNA donor and can template...pathways: classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at...

  7. Plasmids 101: Repressible Promoters

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    Blog Post
    ...created the tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional activation... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...its native context, the tetracycline repressor (TetR) can bind to the tetracycline operator sequences (TetO...(TetO), preventing transcription. In the presence of tetracycline (Tet), TetR preferentially binds Tet...Response Element (TRE)), allowing transcription to proceed. When tetracycline or one of its derivatives is ...other. For example, the GAL4 transcription factor cannot drive transcription from the QUAS promoter, and...

  8. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    ...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...GC-rich hairpin in the RNA transcript followed by a weakly bound poly-uracil tract as shown in the figure ...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...terminators in the context of AAV transgene expression levels. Transcription Termination Rho-dependent termination...

  9. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...altering the amount transfected (see Technical Tips). Minigenome replication and transcription In order to replicate...it will terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...plasmid quality and transfection efficiency may vary between labs, initial titration experiments might ...

  10. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...differ from traditional restriction enzyme cloning will be explained in greater detail. Substrate DNA design...   Substrate DNA Design and Generation Whether you use a linear dsDNA or ssDNA substrate depends ...ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA...find out empirically which is the lagging strand. ssDNA substrate is more efficient than dsDNA with a recombination...approaches.    Bacterial strain with integrated defective prophage A number of E. coli strains exist that stably...red phage. One such strain is DY380, which is derived from the DH10B E. coli strain. Several other bacterial...

  11. Plasmids 101: Mammalian Vectors

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    Blog Post
    ...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to create...reagents vary. Plasmid transfection into mammalian cells is fairly straightforward and the resultant cells...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For many...plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian...

  12. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...changing transfection ratios can affect transfection efficiency. For some transgenes, transfection optimization...containing structural genes and a transfer plasmid carrying the transfer gene. By dividing the viral elements... structural elements, envelope, and transfer gene are transfected into a packaging cell line, usually ...enough concentration, or titer, for your experiment. To improve titer, first optimize transfection conditions... lentivirus can be concentrated by a variety of methods such as ultracentrifugation, centrifugation on...volume than with the unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning...preparations without ultracentrifugation. Sci Rep. 2015; 5:13875. PubMed PMID: 26348152. PubMed Central PMCID: PMC4562269...

  13. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    Blog Post
    ...binding to the template strand (strand used by RNA polymerase II for transcription) are more effective for...days. The crRNA and tracrRNA are annealed on a PCR block and the annealed cr:tracrRNA is then mixed with...complex. This complete transfection mix, i.e. csRNP plus ssODN, is then transfected into stem cells. Cells...experiments. Cas9 protein, annealed cr:tracrRNA, and single-stranded donor DNA. The success of the epitope... efficiency, we have recently demonstrated that the two-part cr:tracrRNA system is much more potent than.... Not only is the cr:tracrRNA approach highly efficient, it is also straightforward as the RNA molecules...prepared with an in vitro-transcribed (IVT) sgRNA versus synthetic cr:tracrRNA. Cells that had successfully...

  14. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

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    Blog Post
    ... complicated transition from a clump of cells to a fully differentiated body. In contrast, these dynamic...two-photon intravital microscopy from PLoS Pathogens - Kamenyeva et al, 2015. Cellular trafficking studies... skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...available for tracking certain cell types. Another useful tool for marking and tracking cells is the the... 18261937. PubMed Central PMCID: PMC2390753. 4. Egen, Jackson G., et al. "Intravital imaging reveals limited...PubMed Central PMCID: PMC4772972. 11. Salomonnson, Emma, et al. "Cell-based and in vivo spectral analysis...fluorescently labelled antibodies made it possible to track cells from one organ to another. Thus several labs...

  15. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ...that increase transduction efficiency, none allow for simple and efficient transduction of both the CNS...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were measured...intensity compared to the strains they were derived from. They also transduced a higher number of neurons... Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged... Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S in the PNS. A GFP transgene was packaged...the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in the CNS...introduce a transgene into a specific cell type in the brain, you needed to make a transgenic mouse. Combining...

  16. 27 Hot Plasmids from 2016

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    Blog Post
    ...and synthetic biology have transformed the way scientists create new traits in bacteria and enabled the...addition to nucleotide-based linking strategies, post translational protein fusion would be advantageous...optimization of industrial yeast strains. However, industrial yeast strains are more difficult to genetically...be difficult to transform. To increase the genetic toolbox for industrial yeast strains, the Borodina Lab...lab from the University of Utah developed SapTrap. The SapTrap system uses golden gate assembly to produce...alone. Molecular processes from signal transduction to transport rely on protein-protein interactions and...be taken with common S288c strains since failing to fully complement strains with multiple auxotrophies...

  17. CRISPR-based Genome Editing Systems For Editing in E. coli

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    Blog Post
    ...pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...applied not only in coli K-12 strains, but also in E. coli B and W strains. The optimized plasmid curing...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...stable and high-yielding strain which had 6 times the yield of the industrial strain. Not only is MUCICAT ...pQCascade) with 4 rounds of transformation to complete the N-acetylglucosamine strain library construction in...multicopy integration. (B) Anhydrotetracycline (aTc) induces expression transposases leading to increased integration...

  18. Plasmids 101: Optimizing Plasmid Yields

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    Blog Post
    ...a protein expression strain, we suggest retransforming the plasmid into a strain meant for DNA purification...incubate Check the bacterial strain Differences between E. coli host strains can impact plasmid yield. Check...to include an extra wash step or use a different strain. For most applications, Addgene uses and recommends...Stable to transform and prep high quality DNA that contains these elements. Be aware of endA+ strains Strains...incomplete. Protein expression strains are not good for cloning Bacterial strains adapted for protein purification...sounds like extra work, but it will likely save time in the long run! While the choice of strain will ultimately...antibiotic concentrations will work just fine; however, for low copy plasmids or slower growing strains, a change...

  19. Antibodies 101: Designing Your First Flow Panel

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    Blog Post
    ...reason, we speak of excitation and emission spectra. Both spectra possess clear maxima where excitation and...Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of...Excitation and emission spectra for APC and APC-Cy7. While the excitation spectra of both fluorophores show...Excitation and emission spectra for eGFP and FITC. As these fluorophores' spectra virtually overlap, you...cells in vitro. I transduced the cultured cells with a virus that encodes a transgenic protein for the ...having a separately expressed transduction reporter is that not every transduced cell will necessarily manage...the transgenic protein. We don't know yet how abundantly CD45 will be expressed on the transduced cells...

  20. Exploring Applications of the Bioluminescent HiBiT Tag

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    Blog Post
    ...Addgene! Following extracellular RNA editors Tracking factors that utilize exosomal transport is quite challenging...luciferase activity is compatible with both intra- and extracellular environments, making it effective for ...within double-stranded RNA. They hypothesized that if ADAR1 were carried by extracellular vesicles (EVs...EVs), edited transcripts could endow neighboring cells with pro-oncogenic traits, facilitating metastatic...degradation as well as endocytic trafficking in real time through extracellular-specific and full lytic measurements...the use of HiBiT to track epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase...hampered by transient interactions with co-receptors (like NRP1) and by endocytic trafficking of the receptor...
Showing: 221 - 240 of 1064 results