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We narrowed to 988 results for: Ran

Showing: 241 - 260 of 988 results

  1. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ...loxp pair, and flips the transgene into the correct orientation for transcription. Upon recombination one...end of the transgene. Since loxp and lox2272 can not recombine, this ensures that the transgene is no longer...flank a transgene (homologous pairs) or used different scrambled sequences to flank the transgenes (non-...Rumpel S (2013) Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5...gene expression in a specific cell type rely on transgenic models that express recombinases like Cre or ...recombinase-dependent AAV vectors for selective transgene expression. While this is a powerful experimental...sequence prior to an ATG start codon to prevent transcription in cells where the recombinase was not present...

  2. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...experiments employ transfection, negative, positive, and replicate controls.  Transfection controls: empty...vector and internal control A transfection control measures transfection efficiency and enables observation... allows you to examine if the transfection reagents or the transfection process itself has any cytotoxic...Another type of transfection control is an internal control vector, which measures transfection efficiency....luciferase) that is either co-transfected with the test plasmid or transfected into a separate well of your...due to the original transfection conditions. Now that we have successfully transfected our cells (Figure...

  3. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...optimized your transduction conditions, you’re ready to transduce! Addgene has a transduction protocol you... the pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to ...functional titering and optimize transduction efficiency. Transduction efficiency can vary with titer, ...cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing cells...this post, I’ll walk you through how to prep and transduce your pooled library.  Pooled libraries Scientists...lentiviral production steps before you can start transducing your virus. Let’s start there!     Figure...sequencing to confirm library representation. Transfection of the DNA pooled library and appropriate packaging...

  4. Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies

    Type
    Blog Post
    ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you...you need to combine multiple transgenes in one fly, having the transgenes in different loci allows for...use? Random insertions take longer because you need to determine in which chromosome your transgene has...brief introduction as to how you can engineer new transgenic flies to study your favorite gene (YFG).  Sometimes... is thrillingly new, you might have to make a transgenic fly in order to study it. The process is fairly...plasmids at Addgene Choosing a vector to generate a transgenic fly There are a few important points to consider...genome? Some vectors incorporate into the genome randomly, while others use attP sequence docking sites ...

  5. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš... PuroR enable sorting and selection of transduced cells. Grant Challen’s lab also created constitutive...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...

  6. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...still direct the translation of proteins because they integrate this reverse-transcribed DNA into the host...genome and then use cell machinery for transcription and translation. Here, we will discuss several aspects...worthwhile to generalize the idea of “transduction” to mean any transfer of material from the virus to the...a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would actually...After uncoating, reverse transcription follows the release of the two strands of viral RNA and of the ...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...

  7. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg and Zhang...developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable Elements ... CAST (CRISPR-associated Transposase) both enable gRNA-directed transposition. INTEGRATE consists of four...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP... unlike other organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining...ineffective in bacteria, CRISPR-induced double stranded breaks (DSB) are lethal. Addgene depositor Luciano...

  8. Another Pathway into Cells: iTOP

    Type
    Blog Post
    ...protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may alter protein... (78-79% after two rounds of transduction.) Compared to CCP transduction, iTOP is at least four times ..., and cell death post-transduction is low. Neuronal stem cells transduced with iTOP resulting in...lab suggests that primary cells may be better transduced using only protein. Read on for a description...description of the lab’s iTOP protein-only transduction method and its potential applications to CRISPR/Cas9 genome...genome editing.   Protein-only transduction? Niels Geijsen’s group in Utrecht wanted to pursue a nonviral...nonviral strategy for transducing primary cells. What if, instead of supplying a DNA construct, they could...

  9. The Golden Stopwatch Award: Recognizing Facilitators of Research

    Type
    Blog Post
    ...Addgene Help Center: Tech Transfer Technology Transfer Information Tech Transfer News: A Reintroduction ...Addgene account for Tech Transfer Offices. They’re packed with tech transfer resources (summaries, electronic...question? Refer them to Addgene’s Tech Transfer Team: techtransfer@addgene.org Congratulations to the Golden...stats and strategies with tech transfer offices. Drop us a line at techtransfer@addgene.org. Albert Einstein...requests from Addgene MTA: material transfer agreement Technology Transfer: the exchange of intellectual property...addgene.org identifies the hold-up. Your Material Transfer Agreement is stuck in “pending.” Fear not, there... to make MTA turnaround faster with technology transfer offices (TTOs). The latest of these efforts is...

  10. Save Time and Money by Making Your Own Competent Cells

    Type
    Blog Post
    ...can find the subsequent transformation protocols below: OpenWetWare Transforming chemically competent ...Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of electrocompetent... easily transformed without a heat-shock step. Because of the large number of transformations being performed...that transforms well in other competent cells) to ensure that your cells can be readily transformed. Compare...Want even more tips for transforming your cells? Check out our transformation guide.   ...We perform over 100 transformations each week at Addgene, so we need to be cost and time efficient. We...a little-known reagent for streamlining the transformation step. Read on to find protocols and tips that...

  11. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    ...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present... present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable expression...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...

  12. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ... not appear to mind extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...modular mini-Tn5 vector that can be used to generate random mutagenesis libraries or to insert heterologous...selecting the important elements from existing transposon and vector systems and creating an all-synthetic...validated this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida...family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on to characterize...mitochondrial turnover Mitochondrial health, transport and turnover are important features to be monitored...

  13. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ... could infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal...integration. After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral...protein), Pol (polymerase), Tat (viral transactivator for transcriptional activation from the 5’ LTR) and Rev...capacity of a lentiviral vector but for which transient transgene expression is sufficient. Lentiviral vector...include a large genetic capacity and the ability to transduce both dividing and non-dividing cells. Lentiviral...integrate into the genome, permitting long-term transgene expression, but they could only infect actively...infect non-dividing cells, but without sustained transgene expression. To design a viral vector system that...

  14. Plasmids 101: Shuttle Vectors

    Type
    Blog Post
    ...mammalian analogue of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is...vectors delivered by transformation and transfection and left the business of transduction to our Viral Vectors... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Plasmids 101: Choosing...to retain the vector as cells divide. In transient transfection, the vector remains episomal, meaning it...vector replication at all. In these cases, transient transfection may be sufficient for your needs even without...called transfer vectors. The gene of interest is cloned into a shuttle vector for transfection into producer...has seen before, showing how DNA sequences are translated into amino acids. And I was astonished that the...

  15. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    ...PHP.B has a >40-fold increase in CNS transduction and transduces the majority of astrocytes and neurons... mice whose CNS was transduced by PHP.B but absent in mice who were not transduced by PHP.B. The Deverman...These results were surprising because in vivo transduction of AAV serotypes generally tracks between strains...diverse library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This... an enhanced version of PHP.B, shows similar transduction efficiency, but at a log lower dose than PHP.B...permissive and six as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive...outbred mice (F1) had half the level of CNS PHP.B transduction as their C57BL/6J parent. The second generation...

  16. Pooled Library Amplifications

    Type
    Blog Post
    ...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...contains 5,000 plasmids, your goal for your transformation would be to obtain 100 x 5,000 = 500,000 colonies...likely rely on serial dilutions to estimate transformation efficiency (Figure 2).    Figure 2: ...

  17. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...Reverse transcription of the transgene occurs in the cytoplasm, then the resulting double-stranded DNA transgene.... However, IDLV genomes are transcriptionally active, leading to transient expression of gene products...lentiviral vectors is random, and the integration site will be different for each transduced cell, there’s no...mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your cells to ...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ...second-generation system as an example.  The lentiviral transfer plasmid encodes your insert of interest, flanked... larger protein: PR (protease), RT (reverse transcriptase), and IN (integrase). These viral packaging ...

  18. Tagging Optogenetics and Chemogenetics Receptors: Fluorescent Proteins and Other Options

    Type
    Blog Post
    ... the transgene of interest. In our studies, mCherry interfered with the transport to the membrane of the...the hM4Di might improve the transport of this DREADD to the plasma membrane. The haemagglutinin (HA) tag...the expression of the transgene of interest and its localization in the membrane. The lesson we learned...2013) could help improve the transport of protein of interest the membrane.   Figure 1: Electron... the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...only a small proportion were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic...

  19. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    ...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used. UAS...for in vitro transcription, the promoter drives either the sense OR antisense transcript depending on ...This element is responsible for initiating the transcription of your insert into RNA. In practice, the term...controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role...expressed.      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...

  20. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    ... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org... in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure...Figure 1: Transformation introduces transformation grade plasmids into a bacterial cell. Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...
Showing: 241 - 260 of 988 results