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We narrowed to 263 results for: Cre expression vector

Showing: 221 - 240 of 263 results

  1. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ... library via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been lentiviral...control for many of the effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9...sites should control for the effects of guide expression and dsDNA breaks. Figure 2: Non-targeting...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently...CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design. Unanswered...CRISPR/Cas9 screen design Are non-targeting guides an appropriate control? Many CRISPR screens use non-targeting...

  2. Zebrafish as a Model for Behavior: Swimming into the Optogenetic Spotlight

    Type
    Blog Post
    Published
    July 16, 2019, 12:59 p.m.
    ... channels, such as ChR2 and NpHR, can even be expressed simultaneously and still be individually activated... like spontaneous swimming. To do so, the lab expressed the LiGluR opsin in different subsets of spinal...optogenetics blog posts Read blog posts about viral vectors Subscribe to the Addgene blog Resources on Addgene.org...spinal cord neurons using the Gal4/UAS system. This created fish lines with different groups of neurons that...

  3. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    Published
    Dec. 17, 2020, 1:30 p.m.
    ...to induce the expression of a Cre recombinase contained within the knock-in. Including Cre-mediated marker...kilobases of additional sequence being introduced. Cre-mediated excision of the selective cassette minimizes...experimental manipulation. In some cases, injection of Cre is required, however the development of the self-...modular toolkits build single plasmid targeting vectors that encode the gRNA transcript and the repair ...using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa), visualize specific genomic....114.170423 Schwartz ML, Jorgensen EM (2018) SapTrap vectors for introducing point mutations with unc-119+ selection...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in...

  4. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    Published
    Oct. 11, 2016, 2:30 p.m.
    ...drive gRNA expression is dependent upon the cell type in question. In plant cells, gRNA expression is achieved...Cas9 expression is commonly driven by plant-derived RNA pol II promoters which regulate expression of longer...mRNAs for gene expression). Examples of commonly used RNA pol II promoters for Cas9 expression include the...carries several useful CRISPR toolkits for creating plant expression plasmids, including plant CRISPR plasmids...system in plant cells. Like other model systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter...OsU6 or OsU3, which are commonly used to drive expression of small RNAs in their respective species. Addgene...with other model systems, multiple gRNAs can be expressed to modify several genomic loci at once (get more...

  5. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette. The target sgRNA can...a) Scheme of validation for DSB mediated EGFP expression cassette reconstitution. When the target sequence...annealing) took place and reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid and ... PX330 plasmids (or similar plasmids for gRNA expression), one gRNA at a time. After 48 hours of incubation...homology directed repair, and resulting in the expression of EGFP. Using this technique, you can assess...clone the PCR products into a pBluescript cloning vector and sequence the resulting plasmid. It can take...mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA." Scientific reports...

  6. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    Published
    Oct. 4, 2018, 12:44 p.m.
    ...tracrRNA was combined with the sgRNA expression vector allowing the screen to be conducted with only two constructs...gain of function screens. These screens can help reveal the function of lowly expressed genes or genes ...regulate gene expression. Some CRISPR libraries contain many sgRNAs per gene to increase confidence in... sgRNA library In addition to inhibiting gene expression, dCas9 can also be used for transcriptional activation...protein-tagging system for signal amplification in gene expression and fluorescence imaging." Cell 159.3 (2014):...Calabrese library to screens performed with open reading frame (ORF) overexpression libraries for resistance...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources...

  7. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    Published
    Aug. 31, 2023, 1 p.m.
    .... They started by delivering an AAV construct expressing channelrhodopsin 2 (ChR2), a blue light sensitive...potential afterwards. This measurement trended with expression of the opsin in the target neurons, validating...studies Channel rhodopsins for optogenetics Viral Vectors 101: Chemogenetics ... intestines known as neuropods. They observed increased vagus nerve firing in response to both of these...

  8. Supporting Rare Disease Research with the 2020 BeHEARD Awards

    Type
    Blog Post
    Published
    Sept. 8, 2020, 2 p.m.
    ... al., 2016). Using lentiviral vectors from Addgene, the lab will express these candidate genes in TS iPSCs... It’s possible that solute channels typically expressed on other cell types can compensate for or bypass...defects. Eight genes from the X chromosome are expressed at significantly reduced levels in women with ... Association for Creatine Deficiencies: Compensating for SLC6A8 mutations in Creatine Transporter Deficiency...SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD) is a parent-let...regeneration of ATP from ADP is creatine. In order to reach the brain, creatine needs to be transported from...Deficiency Creatine Transporter Deficiency (CTD) is a rare X-linked inherited neurometabolic condition and...

  9. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ...in therapeutics, as it can increase gene expression at certain loci to create changes in phenotypes. For...activate, we would recommend using a cDNA overexpression vector rather than going through all the troubleshooting...: once the environmental trigger leaves, gene expression is no longer activated. However, there are several...up-regulated. For instance, generally, the more highly expressed a gene is under native conditions, the less activation...libraries, which consist of plasmids that over-express coding sequences from a given cell type or organism... for altering DNA sequence or regulating gene expression.     References Behan FM, Iorio F, Picco G, Gonçalves...Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging. Cell 159:635–646 . ...

  10. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...subset of RSMs that regulate gene expression) that uniquely express selectable and/or sortable reporters...programs that specify which genes should be expressed or not expressed in each state. To help researchers adapt...register”. Each chemical input to an RSM drives expression of a distinct large serine recombinase that recognizes...system), these reactions are irreversible. Once expression of a particular recombinase is triggered, its...possible downstream states that can be achieved by expression of other recombinases. Multiple orthogonal attB-attP...present a two-input, five-state GRSM register that expresses a unique combination of red, blue, and green fluorescent...state detection with flow cytometry. However, expressing reporters will consume cellular resources and...

  11. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    Published
    May 22, 2015, 4:58 p.m.
    ...avoid accidentally creating a gene drive is also the easiest: don't use a DNA vector that encodes both ...spread exclusively through populations that already express Cas9 from an unlinked locus, while a Cas9+sgRNA...attempting to build gene drives in tropical mosquito vectors of diseases such as malaria and dengue are currently...Cas9 should be aware of the potential hazards of creating “gene drives” capable of spreading through wild...does making transgenic organisms with Cas9 risk creating a gene drive? The simplest way to make a gene ...appropriate flanking homology into a germline cell can create an RNA-guided gene drive. In principle, this event...

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

    Type
    Blog Post
    Published
    Aug. 15, 2017, 1:24 p.m.
    ...loxP sites such that their expression is turned on or off in cells expressing Cre. The result of a system...15 years efforts have been made to create transgenic mice expressing fluorescent protein reporter constructs...allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes...above depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after...useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...respectively. The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable...

  13. 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 ... closed RNAs seem to be conserved in mammals, expressed in neurons, and are especially abundant in neuronal...resulted in a cell migration defect. They have created a circRNA website so scientists can view public...developments. Using the Tet-on system, his lab has created a tetracycline cas9 inducible knock-out system....

  14. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    Published
    Feb. 10, 2016, 3:30 p.m.
    ...is then digested and ligated into an appropriate vector. Gibson Assembly, GeneArt Cloning, and SLIC can...of recombineering. In recombineering, bacteria express phage recombination machinery, which facilitates...products are directly transformed into bacteria expressing recombineering proteins Red/ET from a temperature-sensitive...harnesses the power of bacterial recombineering to create insertions, deletions, and substitutions - at the...

  15. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    Published
    Jan. 19, 2021, 2:15 p.m.
    ...stability. Additionally, the choice of vector can impact the expression level of an antibody (Ayyar et al....such as parasites or microbes, or to proteins expressed on the surface of cells that have been infected...the protein out of solution, or isolate cells expressing this protein. Read on to learn more about antibodies...context of the immune system, each isotype is expressed at different times of the immune response and ...qualitative and quantitative measurement of protein expression in cell lysates, whole cells, or tissue samples...frequently used to compare relative levels of protein expression between cell types or treatment conditions. They....    Figure 4: Western blot showing GFP expression in cells that have been transfected with a plasmid...

  16. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    Published
    Sept. 6, 2016, 2:30 p.m.
    ...consist of a group of genes/proteins that, when expressed, normally encourage cell survival, growth and ...Clone Collection 2.0. The collection is a set of vectors for use with the Gateway Cloning Platform (Life... is that it enables the simple construction of vectors to study the function of any of these genes through...as 30% of all cancers, especially those of the pancreas and colon. Despite the prevalence of Ras mutations...he or she can use to study it (as seen in the screenshot below). Currently, the page includes interactive... Tackling Cancers' Drug Resistance with a New Screening Kit Learn How the Clon Tracer Library Can Be Used...

  17. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    Published
    June 18, 2020, 1:15 p.m.
    ...the phage display screens are subcloned (using FX cloning) into the expression vector pSb-Init, which attaches...their small size, nanobodies can be synthetically expressed off of plasmids in bacteria, and they can target...sybodies can be subcloned into pBXNPH3 or pBXNPHM3 expression plasmids for the production of tag-free proteins...to, like ones that are hidden in the molecular crevices proteins on the cell surface. Figure 1: ...threonines at positions that can be randomized for creating the libraries (Zimmermann et al., 2018). Find ...protein (sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...the candidate sybodies in their coat protein to screen for binding to the target protein. The protocol...

  18. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...flow cytometry, and mRNA expression.   If your protein of interest is expressed at similar levels in ...useful to produce a sample with mixed expression, with the expression measurable through an epitope tag such... sets, you can increase confidence that the labeling observed represents the expression level of the target...epitopes. Detection of overexpressed (tagged or untagged) protein When expression plasmids are available...differences between endogenous expression and transfected overexpression, this approach cannot confirm ...caused by overexpression. Additionally, if the cell line used has endogenous levels of expression, this can...preferred. The overexpression approach is therefore best suited to cell lines without any expression of the target...

  19. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    Published
    Jan. 26, 2016, 3:30 p.m.
    ...that the percentage of dystrophin-expressing muscle cells increases over time, and all three groups confirm...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...confirm dystrophin expression via Western blot, at <10% of the wild-type level. Muscle histology is improved...stem cells 2. Remove any immunogenicity of the AAV vector Once delivery has been optimized, it’s important... reduce off-target editing. Short-term CRISPR expression would be especially desirable, as it would reduce... genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science...

  20. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...marvel of DNA compaction, an enhancer can increase the expression of a gene that is millions of base pairs...genome math expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt ...CRISPR screen. We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an...Putting it all together, we get the following expression for step 1: (H3K4me1 + H3K27ac) – H3K4me3 The...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG enhancers 3. CRISPR...you’re interested in designing your own CRISPR screens for enhancers, check out the Appendix. Conclusion...
Showing: 221 - 240 of 263 results