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We narrowed to 111 results for: cas9 expression vector

Showing: 81 - 100 of 111 results

  1. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    Published
    Sept. 1, 2020, 1:15 p.m.
    ...ideal split vector system. Overall the efficiency of transgene expression from split vectors has been inconsistent... overviews of three split vector strategies plus one additional split vector strategy for increasing the...homology directed repairs (HDR). Split AAV vector approaches Split vector approaches increase the size of the...Generally an overlapping split vector has equal or higher transgene expression when compared to using a single...copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping vector with 440 or 1000bp...dystrophy. While mini-dystrophin expression levels were lower than expression of wild-type dystrophin in normal...to deliver LacZ to muscle in mice. Expression from the hybrid vector, as measured by β-galactosidase activity...

  2. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    Published
    Oct. 20, 2022, 1:15 p.m.
    ...gRNAs A major drawback of CRISPR-Cas9 technology is that most expression systems are constantly “on”. There...surprise then that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same...still effectively serving their function of guiding Cas9 to generate a targeted break. These advances in ...modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA which is part ...trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack...partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In...modifications listed above were developed before CRISPR-Cas9, for use in applications like siRNA and antisense...

  3. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    Published
    Dec. 1, 2015, 3:30 p.m.
    ...using bacterial phage vectors with CRISPR/cas9 sensitizing gene cassettes. Thse vectors are engineered to ... tetracycline cas9 inducible knock-out system. His lab is also exploring the use of cas9 proteins from... closed RNAs seem to be conserved in mammals, expressed in neurons, and are especially abundant in neuronal...There are many clever ways labs are using CRISPR/cas9 and more are being reported. Zhou Songyang from ...

  4. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...(tTA, also expressed by your transfer vector) binds to the TRE and activates the expression of your gene...your experiment using the lentiviral vector system. Viral vectors have been increasingly popular in fundamental...primary cells. Amongst the different vectors used, lentiviral vector constructs have proven very useful ...properties make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components, and fluorescent...fluorescent sensors. Retroviral vector background Until the development of viral vectors, gene transfer into mammalian...un-transfectable cells, labs use viral vectors and particularly retroviral vectors because they target a broader...attractive choice for a gene transfer vector. Lentiviral vectors Although retroviruses are still in use...

  5. Addgene-Seeding Labs Plasmid Grant: Accelerating Science Globally - 5 Scientists, 4 Countries & 3 Continents!

    Type
    Blog Post
    Published
    July 18, 2017, 12:41 p.m.
    ...the light‐inducible bacterial expression system pDawn to clone and express newly identified proteins and...project the expression of isolated bacterial proteins can be achieved using a bacterial expression system ...dietary supplements! Development of a bacterial expression system for renewable biotechnology products Dr...plant, Dr. Robertson aims to make use of the CRISPR/Cas9 technology to engineer plants that are able to destroy...focuses on molecular biotechnology for improving expression of nutritionally and therapeutically significant...cannot produce them. By using yeast integration vectors (see for example this yeast cloning kit) she intends...system such as the pET GFP LIC cloning vector and the pOKD plasmids. The recombinant Cry proteins will then...

  6. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    Published
    June 14, 2018, 11:44 a.m.
    ...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...Three generations of adenoviral vectors: the first generation vectors are deficient in the E1 and E3 regions... second generation vectors are deficient in E1, E2, E3, and E4. Gutless Ad vectors (helper-dependent (...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...death of transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of ...Adenovirus type 5. Three generations of adenoviral vectors The adenovirus virion is a non-enveloped particle...capacity is limited to 8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response...

  7. Tips for arabidopsis transformation

    Type
    Blog Post
    Published
    Oct. 25, 2018, 1:23 p.m.
    ...Addgene  Choosing an expression system Choosing an appropriate expression vector is the first step in .... (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs ...from the Tsuyoshi Nakagawa lab are binary gateway vectors and can be used for promoter swapping and generating...for visual selection of transformants based on expression of RFP in the seed (Emami, Yee and Dinneny, 2013...many insertions can cause artifacts from over-expression and it is likely that such a construct will be... ‘A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology’, Frontiers in Plant...sweet potato contains Agrobacterium T-DNAs with expressed genes: An example of a naturally transgenic food...

  8. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    Published
    Sept. 7, 2017, 12:17 p.m.
    ...Fluorescent Protein Plasmids Find Mammalian Expression Vectors ...that overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate... enzyme assays to select both over- and under-expressing clones. If GFP-encoding cDNA is co-transfected...well plate for further analysis. To assess CRISPR/Cas9 knockout efficacy, we have used both Western blotting...and PCR, depending on the level of endogenous expression. We highly recommend the use of an electronic...ampules each of 4 clones with different levels of expression of our gene of interest. Advantages of the agarose...isolate individual cell clones with different expression characteristics from a population of transfected...

  9. AAV Q&A with Tim Miles

    Type
    Blog Post
    Published
    May 17, 2022, 1:15 p.m.
    ...This will be highly dependent on vector dose. The color diversity in Vector Assisted Spectral Tracing (VAST...drop in transduction coverage compared to just one vector.   Q2: Do you think it is possible to make an efficient...this scenario, you may be best served by a dual vector, split intein approach.   Q3: What’s the best method...for model organisms.    Q11: How consistent is expression is when injecting CAP-B10 or CAP-B22 in marmosets...primer for beginners Adenoviral delivery of CRISPR/Cas9 Tips for a first time AAV user Resources on Addgene.org...Plasmids Adenovirus Guide Page Subscribe to Viral Vectors Blog Posts ...

  10. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...screens Differentially expressed genes  As the costs for global gene expression analyses like RNAseq are...test which of the differentially expressed genes found in a gene expression dataset are causal for a phenotype...differentially expressed genes that are known drug targets, or all differentially expressed genes that code...sequencing to identify which gRNAs are differentially expressed in the separate groups and, as a consequence, ...CRISPR screens designed to target differentially expressed genes can be the next stage in making sense of...interactions to a large extent involve receptors expressed on the cell surface. Thus, a screen can be used...The gRNA oligos are subsequently cloned into your vector of choice using the adapter sequences that you ...

  11. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    Published
    Aug. 7, 2018, 12:31 p.m.
    ... While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges... a usable titer, or fine-tuning selection and expression in your target cell line, researchers often find...transcriptional activation system to drive high levels of expression of certain viral components; according the manufacturer...single 72h harvest is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection...determination of the packaging limit of lentiviral vectors. Hum Gene Ther. 2001 Oct 10;12(15):1893-905. PubMed...Tips for titering your lentivirus Visit the viral vector featured topic page Resources on Addgene.org ...Guide Find lentivirus plasmids Browse all viral vectors ...

  12. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    Published
    Jan. 26, 2016, 3:30 p.m.
    ...long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should be explored to reduce off-target...myoblasts; when injected into DMD mice, these cells expressed functional dystrophin. Editing genes in vivo is..., the CRISPR components would only need to be expressed for a short period of time, which would prevent...et al. show that the percentage of dystrophin-expressing muscle cells increases over time, and all three...three groups confirm dystrophin expression via Western blot, at <10% of the wild-type level. Muscle histology...stem cells 2. Remove any immunogenicity of the AAV vector Once delivery has been optimized, it’s important...off-target editing. Short-term CRISPR expression would be especially desirable, as it would reduce the...

  13. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    Published
    Jan. 18, 2018, 2:34 p.m.
    ...inducible system is found in many bacterial expression vectors. Negative inducible promoter pBad is another...gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant...Temperature inducible promoters Temperature sensitive expression systems are typically less leaky than chemically...chemically induced promoters; they show near-zero expression at regular temperatures but can be induced by ... promoters, in which a gene of choice is only expressed following exposure to a brief heat shock. In the...depositors have developed heat shock-inducible Cre and Cas9 for easy genome engineering in species like C. elegans...promoters Light is another way to activate gene expression, and two-component systems used in synthetic ...

  14. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    Published
    Sept. 21, 2021, 1:15 p.m.
    ...of robust expression of high G+C content genes, as validated through monitoring the expression of fluorescent...different vectors and flow cytometry is used to sort populations by mCherry and GFP expression to check...end-point sequencing assays. These plasmids use “Cas9-mediated adenosine transient reporter for editing... be seen through the success of the change in expression of GFP, for the stop codon should have changed...

  15. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ...directing CRISPR/Cas9 to a targeted genomic location, while shRNAs are used to knockdown expression of mRNAs ...the vector backbone you cloned your insert into and fill out the appropriate boxes with the vector backbone...backbone manufacturer, vector backbone size, total size of your vector + insert, any modifications you ...insert in E. coli, then this is a Bacterial Expression vector. If your plasmid can be classified in multiple...lentiviral vector to be used in mammalian cells, check Lentiviral and Mammalian Expression). This information...provide any full or partial plasmid sequences and any vector maps. The more sequence data available, the better... was a part of the insert (when cloned into the vector backbone). If your plasmid has more than one insert...

  16. Hot Plasmids - November 2023

    Type
    Blog Post
    Published
    Nov. 14, 2023, 3:35 a.m.
    ...fluorescently labeled. Using lentiviral vectors to stably or inducibly express tagged iPAK4 from different promoters...edits, such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...to reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications...activation events as bands of induced GFP-iPAK4 expressed under promoters like the tet-responsive element...to create a three-dimensional atlas of protein expression in the adult mouse brain using thick-section ...

  17. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    Published
    Feb. 8, 2024, 2:15 p.m.
    ...blog CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) CRISPR...cleave DNA and instead activates/represses gene expression by various mechanisms. The RNA guide is typically...- GUIDE-Seq and integrase-defective lentiviral vectors - which rely on insertion of DNA by the NHEJ pathway...Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023 Mar ...specific variants. HypaCas9, sSpCas9(1.1), SpCas9HF1, and evoCas9 are a few spCas9s which have been evolved...of Maya Kostman. The most commonly used Cas, spCas9, has gone through multiple rounds of optimization...

  18. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ...Edit Collection The CRISPR Guide Empty gRNA Expression Vectors...2023). This method uses a prime editor (typically a Cas9 H840A nickase fused to a reverse transcriptase) ...Steensel, B. (2021). Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance...Yoon, S., & Kim, H. H. (2019). SpCas9 activity prediction by DeepSpCas9, a deep learning–based model with... and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019). PRIDICT saves...

  19. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...transgene. Typically, the desired target of RNAi is expressed as inverted repeats separated by a loop region...considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state of the dsRNA... easiest approach to induce RNAi involves the expression of long hairpin RNA (hpRNA). This method is facilitated...construct has unique features, such as inducible expression of the hpRNA and/or fusing your gene target sequence... Although downstream work is required, several vectors can be found at Addgene that could serve as an ...an excellent start (#22988, #137884). Both vectors allow the creation of amiRNAs based upon miRNA precursors...Bassik MC (2016) Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat Biotechnol...

  20. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ...CRISPR-Cas9 genome editing may be the hot new way to manipulate gene expression, but other gene manipulation...recombination, RFP is expressed. Cre can mediate one of two deletions to allow YFP or M-CFP expression; these deletions...ways to spatially and temporally control gene expression, especially in in vivo models, and new Cre-lox...fluorescent protein in the construct, OFP (orange), is expressed when Cre is absent; stochastic Cre excision results... and inversion. The Brainbow-2.1 construct can express one of four colors (n-GFP, RFP, YFP or M-CFP.) ...remaining cassette can invert as long as Cre remains expressed; once Cre is removed from the system, the fluorescent...coding sequence is closest to the promoter will be expressed. This system differs from Brainbow-1.0 in that...
Showing: 81 - 100 of 111 results