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We narrowed to 876 results for: tra

Showing: 281 - 300 of 876 results

  1. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery...on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to daughter strands of DNA....Mattei AL et al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. Nat Commun 9, 597. https...specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started to work...by distinct proteins and result in unclear transcriptional consequences (Sprujit 2013). Enzymatic domains...reality, editing your target site is not as straightforward as expected. TET1 can have many effects independent...

  2. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...into… Transcription units (TUs), which are functional units composed of multiple parts transcribed together...a protein that regulated its own transcription, or the transcription of another gene? Now you have a genetic...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you...Modular cloning, or MoClo, is a systematic cloning strategy designed by synthetic biologists to perform large...hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs. Let’s start...start by examining individual parts and transcriptional units. Individual parts are defined DNA sequences...assembled into larger functional units which are transcribed together. Image courtesy of Robert Hurt. ...

  3. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ...remove one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA ...activity or their translation disrupted through physical blocking of the translation machinery. In the...WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to... by T7 promoters to allow for bidirectional transcription to create dsRNA. Students can produce DNA templates...the temporal and spatial localization of mRNA transcripts in tissue. This is useful for labeling specific...these constructs, developmental biologists have to extract RNA from their organism of interest, use the RNA... gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating Gene function by RNA...

  4. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ... modified cells are transferred into an animal and the behavior of the transferred cells is followed. ...generating a massive amount of descriptive transcriptomics data. Custom CRISPR screens designed to target...pathway with many receptors, signaling molecules, transcription factors, and ligands. The presenter then showed...are lucky and find that a druggable gene has a central role in your system, you could majorly accelerate...smaller validation screen using selected gRNAs extracted from the same or another library. These different...in animal model systems. Use Green Listed to extract gRNAs that target your list of selected genes. ... design. With Green Listed you can also easily extract gRNAs for the same gene from several libraries,...

  5. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    Published
    Feb. 11, 2025, 2:15 p.m.
    ...non-target strand. The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA ...cleaving the ssDNA strands. In this way, Cas3 eats away at both the non-target and target strands by making cuts...every so often. Cutting both strands ensures that there isn’t a template strand left behind that the cell... regions on the non-target strand. Occasional cleaving of the target strand is not as well understood ...bypassed by leaving out Cas3 — a tactic some CRISPR transposase systems utilize with type I-F Cascades. ... section for details). The newly formed single-stranded DNA (ssDNA) is then pulled through Cas3. After...implies — after nicking, Cas3 reels in the non-target strand of DNA much like a fisherman would reel in a big...

  6. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...three double stranded breaks are repaired by two rounds of MMEJ. This double MMEJ strategy looks very similar...genome edits. But have you heard of another double-stranded break repair mechanism: MMEJ (microhomology-mediated...primary methods for repairing DNA after a double-stranded break. HDR copies the sequence from a repair template...fashion, with insertions and deletions common. In contrast, MMEJ uses regions with 5-25 bp of microhomology...are chewed back to reveal homology, allowing the strands to anneal. DNA synthesis then fills in the gaps...5-25 bp) regions of homology flanking a double-stranded break yield precise deletion of the sequence between...homology arms comes more time spent cloning. In contrast, the short homology required by MMEJ can easily...

  7. What Do I Do Now? Academic v. Non-Academic Career Decisions

    Type
    Blog Post
    Published
    March 9, 2017, 3:40 p.m.
    ... area that demonstrate that 85% of leaving postdocs pursue a career outside the traditional academic silo... areas quickly Jack/Jane of all trades More sources of training and resources within the company ...acknowledged perks of scientific and technical training is that these educational paths prepare you for...in US Biomedical workforce will NOT go into “traditional” academic positions. More recently, I have been...encompasses millions of choices including pharma, tech transfer, management consulting, science communication,...perspective on other types of science careers as their training progresses.  In From Academic Solos to Industrial...explicit and implicit aspects of today’s postdoctoral training can directly interfere with a seamless jump into...

  8. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    Published
    Aug. 13, 2024, 1:15 p.m.
    ...column or an ultrafiltration concentrator. As the name suggests, you may also concentrate your prep to...mammalian cells can perform human-like post-translational modifications that bacterial and most yeast ...protein you need to produce.  The most common yeast strain used for recombinant antibody production is Komagataella...be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain...they’ve been separated onto two. You’ll first transfect your cells with your plasmid(s), and then wait...to the desired concentration using these columns. If you are conjugating your antibody in a downstream...Whatever you choose, you’ll want an accurate concentration to give your downstream applications the best...

  9. An Introduction to Adenovirus

    Type
    Blog Post
    Published
    Oct. 3, 2023, 1:15 p.m.
    ... protein translation. Its genome consists of “early” (E) and “late” genes, and the transition between ...makes sense that vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early...sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid for... for any virus, we will see that they are very central determinants of how AdVs behave, and in fact, of...Borkenhagen et al., 2019). AdVs are non-enveloped double-stranded DNA viruses, with a linear genome between 26–45... (and therefore between early and late gene transcription) is demarcated by viral genome replication (...cells that are infected by the vector, only the transgene will be expressed, since the early genes are absent...

  10. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    Published
    July 31, 2018, 12:55 p.m.
    ...and they had been shown to to transduce the retina but only transiently. However new viral vectors capable.... PubMed PMID: 29868583. PubMed Central PMCID: PMC5960719. 8. Trapani I and Auricchio A., 2018. Seeing... years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy...adenovirus (AdV) and lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of...due to its considerable immunogenicity and its transient expression. LV results in long term expression... packaging capacity is well suited for larger transgenes. Unfortunately it has limited tropism in the ...and the one used in Luxturna™, can efficiently transduce various types of retinal neurons. Nowadays a toolbox...

  11. A Career in Grants: How To Become a Grant Professional

    Type
    Blog Post
    Published
    Aug. 20, 2015, 2:30 p.m.
    ...professional development strategy  Build your own Professional Development Strategy to increase your competencies...Approved Trainer by the Grant Professionals Association, and a Certified Social Media Strategist through...the highest likelihood of funding and develop a strategy and projections around that likelihood. We provide... and regulations impacting grant management, we track grant opportunity deadlines, and we ensure that ... materials for publishers. When the community orchestra needed a grant writer, Deb volunteered. Through...consulting firm, a Twitter Chat, and then our grant training and mentoring. How do I become a grant professional...expertise in grant writing, grant management, grant administration, and grant evaluation are available through...

  12. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    Published
    July 8, 2025, 1:15 p.m.
    ... after each transformation and/or use different E. coli strains with higher transformation efficiency....a multigene construct. Figure exemplifies the extraction and use of a coding sequence of interestCDS Level...vector with two other independently regulated transcription units. Created with BioRender.com.   Planning... parts also impacts ligation efficiency, as concentration does not equal the number of DNA molecules. ...solution of a shorter DNA fragment with the same concentration. What really matters is the molar ratio of your...right volume of each plasmid according to their concentration, size, and the desired insert-to-vector ratio...robot, you can set it up to automate the reagent transfer and the restriction–ligation reactions in a 96...

  13. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    Published
    Nov. 12, 2020, 2:15 p.m.
    ...is codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...conformation, folding and stability, change post-translational modification sites and even affect protein function...Mauro and Chappell, 2014). Different rates of translation by different tRNAs, including those that exhibit...actually be critical for determining the rate of translation. The ribosome may slow and pause during elongation...optimization may disrupt the fine-tuned timing of translation and ultimately protein function.  In addition...

  14. Deciphering the Mysteries of Behavior with Viral Vectors

    Type
    Blog Post
    Published
    Sept. 17, 2019, 12:40 p.m.
    ...30644390. PubMed Central PMCID: PMC6333924. Howerton, Alexis R., Alison V. Roland, and Tracy L. Bale. "Dorsal..., inhibiting, or modulating. Viral vectors, in contrast, are considerably more versatile and specific ... circular corridor apparatus using a camera or tracking software. Locomotion is a highly complex process...project to the NAc, where enhanced glutamate transmission leads to locomotion sensitization. (for review... Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC projections to the NAc and decreased...risky). Taking advantage of these characteristic traits, rodents can be used to study anxiety levels in...injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression...

  15. Lab to Office Culture Shock

    Type
    Blog Post
    Published
    Feb. 5, 2019, 1:04 p.m.
    ...opportunity to discover and evolve your transferable skills. Transferable skills are the non-technical skills...employee who's coming directly from grad school. The transition has been a bit rough for her (3 weeks in and ...there are any resources that can help with the transition.” The lab-to-desk-job culture shock is a real...scientist colleagues have as well. Making the transition from the bench can be jarring for the unprepared...resources out there for scientists making this transition. In this installment of the Addgene career guide...formulate hypotheses and conduct experiments. You transfer liquids between tubes, look through microscopes...What should you expect during the lab → office transition? I’ve already provided a few counterpoints to...

  16. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    Published
    June 6, 2023, 1:15 p.m.
    ... Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesize complementary...alternative virus, or other strategies may need to be considered if size constraints are an issue. Adenovirus...ample room for most experimental needs, and can transduce (infect) a wide range of cell types with a high... such as a heterologous envelope or a post-transcriptional regulatory element can be used for either type...Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some...replicate episomally, essentially remaining an extrachromosomal part of the host genome that can replicate...response as retroviruses. This virus can also transduce dividing and non-dividing cells, making it conducive...

  17. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

    Type
    Blog Post
    Published
    July 28, 2015, 2:30 p.m.
    ... pair has focused considerable attention on transcription factors, starting with an established method... relies on antibodies to recognize and bind transcription factors directly, but that’s a slow-going process... it doesn’t work in many cases. In an effort to track down every last one of those gene regulatory proteins...ENCODE project is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the... ChIP-seq, but it’s really hard for lots of transcription factors. Our groups got together to adapt CRISPR...big difference. We can now look at many more transcription factors. Addgene: How does the system work? ...second plasmid to target an epitope tag onto the transcription factors. By the way, you can actually use this...

  18. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ... was on the right track. The next insight was to assemble a synthetic transcription activation complex...after natural transcription activation processes. Konermann and her colleagues demonstrated the new system... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...each transcription start site in those 15,977 human genes. Weissman and his colleagues demonstrated in ...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2-P65-HSF1 plasmid which...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...

  19. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    Published
    Nov. 3, 2020, 2:15 p.m.
    ...membrane localization of transmembrane proteins, like rhodopsins, are highly constrained and poorly understood...delivered ChRgers by intravenous injection of PHP.eB, an AAV capsid capable of broad transduction of the nervous... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one region of the...channelrhodopsin, such as cell localization, kinetics, and spectral properties, must be optimized simultaneously in...channelrhodopsins with desired characteristics. The model was trained to predict if a channelrhodopsin sequence would...neurons and mouse brain slices collected following intracranial injection of an AAV expressing a ChRger. In ...

  20. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

    Type
    Blog Post
    Published
    March 3, 2020, 2:15 p.m.
    ...the co-transduction by two viruses would lower editing efficiency, the split editors transduced at levels...colleagues evaluated CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this...capacity of 4-5 kb limits the delivery of larger transgenes, such as base editors which have a gene size ...base editor is split into two plasmids and co-transfected into cells for reconstitution. Image from Levy...causes Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the split base... of response and brightness. They also do not spectrally separate from green sensors and blue-light-activated...Image from Beck and Gong, 2019. Broadening the spectral diversity of GEVIs can enable resolving single...
Showing: 281 - 300 of 876 results