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We narrowed to 1,064 results for: tra

Showing: 241 - 260 of 1064 results

  1. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ... by the bottom strand versus nicking the bottom strand first followed by the top strand. Both produced...increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2...complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded by uracil...present on both strands of the plasmid. The ssDNA template is generated by transforming a backbone phagemid...ExoI then fully degrades the nicked strand. Synthesize mutant strand: A mix of degenerate primers and the...Degrade wild-type template strand: Next, the opposite wild-type DNA strand is nicked by the BbvCI variant...step 1. Then this strand is degraded with ExoIII and ExoI. Synthesize 2nd mutant strand: The second mutant...

  2. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...from their compartment of action via sequestration. The strategy relies on several observations: Rapamycin...appear to mind extraneous proteins bound to its outer mitochondrial membrane. Sequestration is accomplished...GATEway Cloning Frustrated by the complexity of assembling recombination of transgenesis constructs? A ...the intracellular kinase domains into contact and initiating signaling. Grusch et al. demonstrate that...CRUSH vector in transgenic mice, the lab established a dual-color RNAi “sensor” mouse strain in which Cre...constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation. Addgene...each transgene expresses one of three possible fluorescent proteins, chosen for minimal spectral overlap...

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

    Type
    Blog Post
    ...vectors for selective transgene expression. While this is a powerful experimental strategy, one of the most...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...Vectors). While this strategy greatly reduced the overall level of off-target transgene expression, low levels...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...2020) Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations. Front Synaptic Neurosci...

  4. Tips for Titering Your Lentiviral Preps

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    Blog Post
    ...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used...qPCR based methods for viral titration consider using a reverse transcriptase mutant in a separate control...deficient in reverse transcriptase will produce low levels of virus and can be used to track carryover. Functional... positive for vector encoded transgene expression following transduction with serial dilutions of a viral...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a ...can also impact transgene expression. The most accurate method of lentiviral titration uses qPCR to measure...

  5. Multiplex Genome Editing with CRISPR-Cpf1

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    Blog Post
    ...the usual transfection drawbacks: transient expression and needing to work with a transfectable cell line...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...expression platforms: transfection, lentiviral transduction, and AAV transduction.  In most of Zetsche ... rely on transfection of more than one vector to express the gRNAs and Cas9. Co-transfections can lead... GFP tag were transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of...Lentiviruses carry a (+) strand RNA copy of the DNA sequence, which is a suitable substrate for Cpf1. This means...

  6. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    ...Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine tract; recognition...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...

  7. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    ...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...enhancers and the transgene; such interactions can alter expression of not only the transgene but also that...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...

  8. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...., 2017), axonal transport  (Murlidharan et al., 2014), and postsynaptic transmission (Oh et al., 2020... S. (2013). Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5... an Immune Response against the Tetracycline-Dependent Transactivator Correlates with Long-Term Doxycycline-Regulated...Doxycycline-Regulated Transgene Expression in Nonhuman Primates after Intramuscular Injection of Recombinant...adeno-associated viral vector-mediated transduction of the corticospinal tract: Comparison of four promoters. ...Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer. Molecular Therapy - Nucleic...great in a GABA neuron, for instance, may not transduce the adjacent norepinephrine neuron at all. There...

  9. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...their straightforward husbandry and ability to produce abundant, externally fertilized, transparent progeny...per individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside ...mRNA inhibit its translation either by blocking initiation or splicing. (RNAi, in contrast, engages existing...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...different experimental conditions. Strategies for generating transgenic zebrafish are well established (...Mosimann, 2016). One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign...lineage tracing Over a period of 24 hours, the externally fertilized zebrafish zygote transforms into an...

  10. 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...GFP-expressing Plasmid B, could demonstrate whether the cells were transfected successfully and expressing...

  11. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...species. Existing protocols can transfer CRISPRi into a single bacterial strain, such as a B. subtilis, or ...bacterial Tn7 transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes ...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...: A Tn7 transposon containing the CRISPRi components and a plasmid with the Tn7 transposition genes are...are transferred to a recipient bacteria strain of interest by tri-parental mating. Donor bacteria contain...disease. However, the rate of transfer was variable. They measured transfer rates by comparing the number... P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...

  12. 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...

  13. New Tools Enable CRISPRa for Neuroscience Applications

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    Blog Post
    ...engineered combinations of transcriptional activators to induce gene transcription at genomic sites targeted...p65-HSF1 transcriptional activator to each genome-bound dCas9 (see Figure 1a for the transgenic construct...different untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional start...relatively straightforward to use CRISPRa to selectively target these different transcriptional start sites...distinct transcriptional start sites, all of which yield the same BDNF protein. To demonstrate the specificity...investigation. Co-transduction of primary rat neurons with dCas9-VPR and sgRNAs targeting either transcript variant...are often many more genetic strategies) and then consider whether traditional cDNA overexpression is suited...

  14. Viral Vectors 101: The AAV Retrograde Serotype

    Type
    Blog Post
    ...information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular... either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...making them useful tracers, but finding a virus that works well for retrograde tracing can be difficult....naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at the cost ...expression.     AAV retrograde Unlike traditional dye-based neuronal tracing reagents, AAV particles require...not be transduced at all by this serotype. Nichole Beebe in Brett Schofield’s lab demonstrated that pathways...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...

  15. 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... transgene is incorporated into a large complex of both viral and cellular proteins and trafficked to .... However, IDLV genomes are transcriptionally active, leading to transient expression of gene products...mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your cells to ... for stable expression of the transgene. This makes IDLVs an attractive choice for certain types of clinical...quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and Yáñez-Muñoz...They rely on a viral protein called reverse transcriptase to convert their RNA genomes into DNA to be ...

  16. 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...s lab demonstrated that a minimal, regulatable CRISPR system effectively silenced transcription of one...repressed or activated transcription of select genes in E. coli. Furthermore, to demonstrate wider applicability...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP...

  17. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ...resonance energy transfer (BRET). The fluor then emits light at a tissue-penetrating wavelength above ...and imaged after i.p. or i.v. administration of the furimazine substrate for NanoLuc luciferase. Both ...luminescence. Intracellular Calcium Sensor Yang, J. et al. (7) wished to trigger intracellular Ca++ release...imidazopyrazinone substrate. ACS Chem Biol 7, 1848-57. PubMed PMID: 22894855. PubMed Central PMCID: PMC3501149...cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted...group as well as evolution of the coelenterzine substrate to the more stable yet brighter furimazine by ...can generate autofluorescence and have limited penetration due to absorption by tissues. To avoid absorbance...

  18. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...editing techniques. Inbred and outbred strains Mouse strains used in research can fall under one of two.... Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally ...genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic heterozygosity...population. Outbred strains of mice are useful for comparing dominant and recessive traits and their impact...inducible gene expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain...DNA randomly into the mouse genome. In contrast, knockin strains are generated via homologous recombination...and provides a way to track successfully engineered cells. Similar to transgenic mice, knockout mice are...

  19. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-FPS are better for whole cell imaging...By generating PA-FPs fused to intracellular proteins, scientists can track single protein localization,...localization, turnover, and trafficking, as well as organelle trafficking and dynamics (fission, fusion, etc). For...PubMed Central PMCID:PMC4007248. 11.Hess, Samuel T., Thanu PK Girirajan, and Michael D. Mason. "Ultra-high... proteins that display unique changes in their spectral properties upon exposure to a specific wavelength... wtGFP normally contains a mixed population of neutral (protonated) and anionic (deprotonated) forms of...generates a chromophore population that is more neutral, causing PA-GFP to display almost no fluorescence...

  20. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...structures in RNA transcripts Effects on transcription (remember, it’s not all about translation - e.g. codon...codons encode single amino acids. Evolutionary constraints have molded which codons are used preferentially...in any organism. So what are the evolutionary constraints that lead to these preferences and what can we...therefore may need fewer resources for the entire translation process. For example, during high growth rate... tRNAs. For example Tuller et al. found that translation efficiency is well correlated with codon bias...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...
Showing: 241 - 260 of 1064 results