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We narrowed to 369 results for: Cre expression vector

Showing: 221 - 240 of 369 results

  1. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    ...LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis of bisphenol A-induced perturbation...cytoskeletal disorganization, apoptosis, and gene expression disregulation on HeLa cells." Journal of applied...with the Agarose Method Plasmids 101: Mammalian Vectors Resources on Addgene.org Find mammalian plasmids...plasmids for your research Find viral vectors for infecting mammalian cells ...making their surrounding media acidic through the secretion of lactic acid (a by-product of cellular metabolism...

  2. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-...BFP variant, a BE4am-Cas9 base editor, and sgRNA vectors for the BFP-to-GFP conversion and to target specific...integrated it into human and mouse cells that expressed either Cas9 or an optimized cytidine base editor...antibiotic resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing... of a mutated start codon to initiate protein expression, it can be used for an array of detectable proteins...

  3. Plasmids 101: The Wide World of Natural Plasmids

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    Blog Post
    ...other molecules to host organisms. For instance, vectors derived from the Agrobacterium Ti virulence plasmid...especially when combined with natural inducible expression systems that allow tuning of metabolic pathways...developing new cloning techniques based on these vectors (Shareck et al., 2004). Plasmid categorization..., Y., Lee, B., & Miguez, C. B. (2004). Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic ...Figure 1: Common functions of natural plasmids. Created with BioRender.com. Resistance (R) plasmids...resistance genes to help cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids...years, and new classifications are still being created as whole-genome sequencing becomes more relevant...

  4. Plasmids 101: Origin of Replication

    Type
    Blog Post
    ...plasmids with low to medium copy numbers can still express massive amounts of protein given the proper promoter...the table below for more details.   Common Vectors Copy Number+ ori Incompatibility Group Control ...Relaxed This table defines common cloning vectors, their copy number, ori, and incompatibility groups...R6K, and 15A. Not all origins of replication are created equal. Some will produce many plasmid copies and...because they will compete for the same machinery, creating an unstable and unpredictable environment. As ...plasmids if they contain large inserts or genes that create a toxic product. The E. coli strain Most E....

  5. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

    Type
    Blog Post
    ...use plasmids from Addgene to construct NFIX expression vectors as well as reporters of NFIX function. They...interactions and how these interactions regulation gene expression. Recent studies have shown that the conditions...delete the functional portion of the Nfix gene using Cre recombinase and to generate Nfix-negative neural ...interactions and how these interactions regulation gene expression.     ...burden of immune symptoms on CDG patients and to increase their quality of life by reducing their risks ...ligand binding sites in T4 lysozyme by deficiency-creating substitutions." Journal of molecular biology 277.2...

  6. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    ... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...confirm the presence of an insert in a particular vector by excising the insert from the backbone. To do...will need to know both the approximate size of the vector backbone as well as the predicted size of the insert...time-intensive, so we recommend a variety of ways to screen and verify your plasmids. Here, we'll cover restriction...500bp to 10kb, with the 3.0kb fragment having increased intensity to serve as a reference band. The uncut...

  7. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...My lab's vector of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, ...shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes which help mediate AAV viral ...Production of recombinant adeno-associated viral vectors for in vitro and in vivo use. Current protocols... out what makes AAV a versatile tool for gene expression in mammals Use the PEI calculator for planning...transfer plasmid at 30 °C instead of 37 °C and then screen for ITR recombination with a SmaI or XmaI restriction...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...mL of my AAV transfer plasmid cultures and then screen with SmaI digests. I freeze the bacterial pellet...

  8. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    ...in patched cells expressing wild type CoChR, these APs are absent in soCoChR-expressing cells. Finally,...C-terminus of the protein. Unlike the diffuse neuronal expression observed with wild type CoChR, soCoChR localizes...stimulation are largely absent in the soCoChR-expressing system. Taken together, Shemesh et. al. describe...Pseudomonas putida CBB5 and has been tailored for expression in E. coli. This operon is under the transcriptional...strong constitutive promoter (BBa_J23100) and expresses N-demethylases and other required enzymes, including...Moreover, a caffeine “addicted” E. coli strain expressing this operon can serve as a living biosensor for...FRET reference page for many other common FRET vectors. Murakoshi H, et al. Sci Rep. 2015. Pubmed PMID...

  9. Plasmids 101: Protein tags

    Type
    Blog Post
    ...Fluorescent Protein (GFP) Fluorescent Protein Guide Expression Vectors from the Berkeley QB3 MacroLab Eric thanks...box of tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags...purification to get your protein as pure as possible? Vectors that offer different combinations of tags are readily...Biochemists and molecular biologists who need to overexpress and purify proteins can face any number of technical... some of the hurdles to overcome in order to overexpress a recombinant protein? It is not generally in...in a cell’s best interest to overexpress a protein. Energy and cellular resources are being spent to make...stabilization, as can maltose-binding protein (MBP). Overexpression can also lead to insolubility, and aggregated...

  10. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    ...epitope tags, low FP expression may be insufficient for monitoring a weakly expressed POI. Spaghetti Monster...antibody-based detection, smFPs create much brighter signals for weakly expressed proteins than standard tags...cytotoxicity, low immunogenicity, and the long-term expression of AAV delivered genes. AAV serotypes have been.... Thanks to this work it is now much easier to express molecules in your favorite neuronal population ...insufficient for detection when the POI is weakly expressed. As these epitopes tags have no intrinsic visual... be fused to scaffold proteins in order to be expressed stably in neurons. As compared to epitopes tags...labeling of neurons. As discussed above, weakly expressed proteins in particular are difficult to track ...

  11. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    ...protein of interest into a vector encoding a fluorescent tag. Once expressed as a fluorescent protein fusion...bacterial components. For example, the Rainbow Vectors from the Mariette Barbier Lab can be used to fluorescently...Barbier, Mariette, and F. Heath Damron. "Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling... to its final destination. As an example, most secreted proteins are produced in the endoplasmid reticulum...golgi and then transported in vesicles via the secretory pathway to their subcellular or extracellular ... they are finally degraded (2, 3). Once you've created your fusion protein, it's important to validate...subcellular structure or protein complex. By coexpressing both the marker protein and the protein of interest...

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

    Type
    Blog Post
    ... 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...

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

    Type
    Blog Post
    ... 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...

  14. AAV Production in HEK293 Cells

    Type
    Protocol
    ...Cells You may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference...transfection: pHelper; pRC (Rep-Cap), plasmid expressing your gene of interest Triton X-100 Benzonase/...harvesting. The addition of sorbitol has been shown to increase viral titers by up to 1.8-fold ( Rego et al., ...sodium bicarbonate, sorbitol has been found to increase viral titers by up to 1.8-fold. See the recipe...

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

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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...

  17. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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...

  19. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    .... 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...

  20. Supporting Rare Disease Research with the 2020 BeHEARD Awards

    Type
    Blog Post
    ... 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...
Showing: 221 - 240 of 369 results