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We narrowed to 784 results for: SCR;

Showing: 101 - 150 of 784 results

  1. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...hot plasmids segment on the Addgene Podcast. Subscribe to the Addgene blog to get all the latest hot ...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid N-terminal... did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors...the RFPs developed from natural ancestors), and screened through libraries of new RFP variants for increased...beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl...

  2. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the ...mutations found in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1 galE15 galK16...cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR...General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient; good strain for...derivative. General cloning and storage, blue/white screening. F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74...

  3. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    ...our AAV preparations. We recently published a description of the VGS process and use cases in Human Gene...ligation and tagmentation preparation approaches described previously require a preliminary double stranding...addition, we wrote an open-source custom Python script that uses the VGS data to determine the percent...recombination present in Cre-dependent vectors and a script that can confirm the serotype of AAV preparations...use this to our advantage by designing a Python script that looks for pre-defined serotype specific signature...signature sequences in the data. Unlike previously described thermodynamics-based methods of serotype confirmation...parallel and provide serotype data in minutes and the scripts  can be easily customized to detect other sequences...

  4. Summer SciComm: Preprints

    Type
    Blog Post
    ...is the rising use of preprints, or scientific manuscripts shared by the authors on a public server independent... their work publicly and immediately once the manuscript is drafted. Preprints were one of the key routes...at the same time as, or before, submitting the manuscript to a journal for formal peer review. Some organizations...unpublished results at a conference? Post your working manuscript as a preprint too and you can share all the details... use your work but don’t think they’ll have subscriptions to the specialized journals in your field? Post...only way to rigorously determine or ensure a manuscript’s quality. But even journal-based peer review ...giving authors the opportunity to strengthen the manuscript prior to submission. Preprints at Addgene Preprints...

  5. The Addgene Antibody Data Hub

    Type
    Blog Post
    ...contains a detailed description of the experimental conditions.     Figure 4: A screenshot of the materials...antibody worked under the conditions described above, and a short description of how the antibody performed....the submitted application, along with a brief description of the experiment. If that data was included ...conditions in which the data was generated, you can scroll down the page to the materials and methods section...The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate, and...

  6. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and translation...structural proteins expressed from a reverse-transcribed piece of DNA called a “provirus.” This DNA is...(or host cell) by the viral protein reverse transcriptase (RT). In the case of a natural virus, both the...the protein and the genetic code for reverse transcriptase are packaged (Wu et al., 1997) inside of the...viral vectors, only the protein for reverse transcriptase (RT) is present in the capsid, as the engineered...is a turn of phrase — “pseudotransduction” — to describe the direct protein transfer of RT, which would...during uncoating. After uncoating, reverse transcription follows the release of the two strands of viral...

  7. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    ...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If... population of cells as part of pooled library screens (1,2). The human and mouse GeCKO libraries, targeting...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...you get full library coverage in pooled library screens. Availability and Use LentiCRISPR v2 is available...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...

  8. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    ...methodology of software development called Scrum. The Scrum framework is based on an iterative and collaborative...team because we are using the Scrum framework or we are using the Scrum framework because we are highly...collaborative approach to development. Scrum teams plan and break up work into small, well defined chunks...needs of the teams at Addgene. Another advantage of Scrum is that it provides transparency to everyone involved... on a new feature. We start with a story that describes the feature and what the criteria is for accepting...that the clarifications lead to the story being scrapped or broken up into smaller stories. Since this ...

  9. Let There Be LITE Plasmids

    Type
    Blog Post
    ...of customizable DNA-binding domains based on transcription-activator-like effectors (TALEs) as anchors ... thaliana. The results are light-inducible transcriptional effectors (LITEs) designed to bind specific.... Genes under LITE control showed a rise in transcription in as little as 30 minutes after light stimulation...News & Views Andreas Möglich and Peter Hegemann described several advantages of the new system: "First,... universally abundant. Second, induction of transcription occurs within minutes of light exposure. Third...really flexible and modular,” Konermann said. “Transcriptional activators are easy to expand or combine with...al. "Optical control of mammalian endogenous transcription and epigenetic states." Nature. 500, 472–476...

  10. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    ...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...tagging RNA sequences with an aptamer and RNA transcription by placing the aptamer downstream of a known... Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes...uniquely transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence by flow cytometry... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...Wenjiao, et al. "Imaging RNA polymerase III transcription using a photostable RNA–fluorophore complex....

  11. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ...typically fairly high, so there is usually no need to screen a large amount of colonies. Excluding the time ...combined with the popular CRISPR technology and is described in a recent publication by the Lockey lab (Wang...followed by the standard Gibson assembly techniques described above, this resulted in the direct and seamless...using Gibson Assembly along with CRISPR was also described recently (Jiang et al. 2015), in which very large...challenging. Other homology based technologies We've described Sequence and Ligation Independent Cloning (SLIC...

  12. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    ...from one that represses transcription to one that activates transcription.However, this system lacks spatio-temporal...barbara_synbio) May 13, 2021 The original AraC transcriptional regulator Their system is based on the AraC...AraC protein that controls transcription from the PBAD promoter. “AraC is widely used in synthetic biology...It relies on the sugar arabinose to activate transcription by causing conformation changes in the AraC ...domains of the BLADE dimerize and activates transcription. The team created two constructs, among others...with AraC,” says Di Ventura. BLADE activates transcription with the flick of a light switch and shuts off...

  13. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring...Sabrina Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized...

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

    Type
    Blog Post
    ...the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead ribozyme parts are described in ...of an RSM is defined by the sequence within a prescribed region of DNA termed the “register”. Each chemical...registers within a sample using a metric that we describe in the Materials and Methods section of our paper...sensors do not necessarily have to be based on transcriptional regulation, as long as they can control recombinase...recombinase activity. For example, post-transcriptional recombinase activation via RNA toehold riboregulation...to read states by qPCR or Sanger sequencing as described in the Material and methods section of our paper... Multiple large serine recombinases have been described in the literature (5, 6) and may be substituted...

  15. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    ...lentiviral packaging methods. The three components described above (envelope, packaging, and transfer) are ...nomenclature of the plasmid backbone is meant to be descriptive of the components found within the backbone. ... Pol Precursor protein containing Reverse Transcriptase and Integrase components. Transfer cPPT... WPRE Woodchuck hepatitis virus post‐transcriptional regulatory element; sequence that stimulates...necessary for activation of viral genomic RNA transcription. R; Repeat region found within both the 5' and... 5’ LTR Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R,...rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition of a poly ...

  16. Why and how to start studying chromatin (with Addgene's help)

    Type
    Blog Post
    ...972.e23 Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated...times have you looked at a diagram depicting transcription, or DNA repair, or replication, or any number...researching enhancer elements that control the transcription of a gene a megabase away, or looking at the...plasmids containing the indicated gene, and you can subscribe to these pages to learn when Addgene receives ... articles reporting chromatin-related research describe the production of plasmids that might be useful...colleagues and guest bloggers have written posts describing new methods and tools for studying chromatin ...

  17. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    ... lentiviral vector that contains a scrambled shRNA. This scrambled shRNA is used as a negative control...sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP ...virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.(If you’ve peeked...envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini’s lab...control for mammalian RNAi experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This...the necessary elements for packaging, reverse transcription, and integration needed for the production of...

  18. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, they can deplete...that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The system...tetracycline or one of its analogs will induce these transcriptional changes, and expression is tunable by titration...expression goes back to baseline) and occurs at the transcriptional level.     Fig. 2 Tetracycline...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

  19. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    ...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and... of xCas9 3.6 and 3.7 shows that we have only scratched the surface of modifications that can be made ...

  20. Pooled Library Amplifications

    Type
    Blog Post
    ...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab...library has inserts of different sizes (such as a transcription factor overexpression library) or is prone to...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...et al. Nat Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...More resources on the Addgene blog Genome-Wide Screening Using CRISPR Preparing Pooled Libraries NGS Quality...

  21. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    ...open reading frame (ORF) library to screen PPIs among 450 transcription factors in live fruit flies. Although..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...large number of constructs for high-throughput screening of PPIs. For example, Bischof et al. (2018) generated...organisms lags behind. BiFC can also be used to screen for small-molecule inhibitors of a particular PPI...quantification (Kerppola, 2006, 2013). These are described in more detail below: Fusion protein construction...

  22. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    ...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ... of the book, called simply “Old Science,” he describes the now infamous tale of Rosalind Franklin and...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...This last section of the book, Future Science, describes the author’s thoughts on science’s biggest challenges... doesn’t need to. The problems and challenges described in The State of Science have not changed in “COVID...

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

    Type
    Blog Post
    ...CRISPR screen to disrupt these enhancers. The third and final step is to conduct our CRISPR screen. We start...stem cells, and conclude with a targeted CRISPR screen that can disrupt these enhancers. A real-world ...“GPS for proteins”) for NANOG, an essential transcription factor in embryonic stem cells (ESCs). Your ...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...established with Bowtie2 using the parameters first described in Kearns et al.: bowtie2 -f -x HG19_GENOME --...

  24. "Hall of Fame" AAV Enhancers from the Allen Institute for Brain Science

    Type
    Blog Post
    ...expression by interacting with proteins such as transcription factors and chromatin modifiers. These proteins...at the promoter region of a gene to initiate transcription (Figure 1). Although all cell types share the...functional in every cell. Differential expression of transcription factors, which recognize and bind to different...capitalized on. Figure 1: Schematic of transcriptional regulation by an enhancer. Created with BioRender.com...includes annotated images of the brain sections describing the labeled regions, as well as the strength ...just that, and their painstaking efforts towards screening thousands of enhancer sequences has culminated...nrg3458 Ray-Jones, H., & Spivakov, M. (2021). Transcriptional enhancers and their communication with gene...

  25. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...after the piñata burst, value was subjectively ascribed relative to the basic needs of each individual... few prior publications to go on, a submitted manuscript could only have been viewed as a significant ...There are 8 major categories found in the table, describing everything from basic to advanced functionality...the user is expected to interact with the tool (a screen shot and overview of the table can be found above...Software design elements that fall into this category describe how the user is expected to interact with the ...require working knowledge of the command line or scripting. Input flexibility: Software tools require some...re engaged in heavy CRISPR design (I’m talking screens and/or very large genomes), then it is worthwhile...

  26. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ...reverse transcription. This is then amplified by PCR using Taq Polymerase and then transcribed to provide...Cas12a activity gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied...amplified. RPA reactions also contain reverse transcriptase to produce cDNA. Isothermal amplification is...thermocyclers. This is followed by in vitro transcription and Cas13 is then used to detect the amplified...designed, each binding to one strand of the reverse-transcribed target sequence that are present close to the...fluorescence upon cleavage. Highlights: Reverse-transcribed RNA samples are amplified by PCR instead of RPA...

  27. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we were very excited...find the 0.2-0.5% correctly edited alleles, when screening reasonable amounts of single cell clones. Furthermore... together with the intended sequence change and screen a few hundred clones to find the right one Step... few hundred. From these two steps, you’ll be screening a total of a few hundred clones - Still better...longer feasible to incorporate mutations without screening thousands of clones to find a positive one. So...thinking further about the distance relationship described above, we realized that, in addition to increasing... and neurovascular diseases. He worked on the described project while he was a Postdoc in Marc Tessier-Lavigne...

  28. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ... They use a unique and powerful high-throughput screen to select variants based on performance metrics...: JEDI-2P was identified from a high-throughput screen, selected for increased voltage response, brightness...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization of...

  29. Cellosaurus: A Cell Line Information Database

    Type
    Blog Post
    ...database describing nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all ... resource.  This is a labor-intensive project; scripts can be used in some cases, but much of this work...authors of the paper in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by...information about a cell line you’ve used? Submit your manuscript or data to Cellosaurus via [email protected] received a report that HT-55, which was described as a colon cancer cell line, is actually a rectal...

  30. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
    ...have used PCR-based screening to detect mutants, but this method limits screening to a few hundred worms... the summer of 2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C....can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just...knock-ins. These candidate animals can then be further screened based on their Roller phenotype. If SEC is inserted...wild-type animals that have undergone SEC excision are screened based on the loss of the Roller phenotype. These...

  31. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ... have been generally found to have a higher discrimination at the upstream side of the ligation junction...including Taq DNA ligase, are naturally able to discriminate against ligating substrates containing base ... at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature... 27). This methodology has been used to rapidly screen buffer conditions for Taq DNA Ligase and their ...electrophoresis (CE) (see below). This method allowed screening of all possible base combinations (Watson-Crick...of a 96-well plate, allowing 6 conditions to be screened per plate. The results indicated that the optimal...optimization method has been used internally at NEB to screen additional ligases, conditions, and formulations...

  32. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...Zhang and is described in Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE,...is bad news for repeatability and bad news for a screen. Because Addgene viruses are provided at a specific...expression cell line. Especially if you’re doing a screen. I made a few monoclonal lines (Figure 2) and I...was a gift from John Doench & David Root and is described in Rational design of highly active sgRNAs for...were gifts from John Doench & David Root and are described in Optimized sgRNA design to maximize activity...Additional Resources on the Addgene Blog Genome-wide Screening Using CRISPR/Cas9 Deliver Multiple gRNAs at Once...

  33. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    ...analysis scripts available, some associated with specific libraries, but any analysis script should return...plasmid. However, lots of library DNA is needed for screening experiments, so the scale of the DNA prep is larger... mixture on solid media. The colonies are then scraped off the plates and pelleted for DNA extraction....automated manner. Addgene commonly uses a custom script derived from the count_spacers.py file from Feng...wishes you luck in your future library use and screening, and if you have any tips, input, or stories of...

  34. Addgene Welcomes John Doench to the Advisory Board

    Type
    Blog Post
    ... primary screen to knowing what are the real hits is so much shorter…. it’s a great screening technology...researchers in a variety of fields develop RNAi screens to answer their own research questions. Starting... For his post-doc, Doench worked on using RNAi screens to learn more about cancer biology in Ed Harlow...developing CRISPR pooled libraries for use in genetic screens and has contributed much of his knowledge to the...become more efficient in terms of editing and screening) such that thousands of individual edits can be...

  35. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    ...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher...worries, I've summarized the three strategies described in this post in the graphic below - including ...

  36. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...system is much more potent than the in vitro-transcribed single guide RNA (IVT sgRNA) approach (Dewari...comparison of Cas9 RNPs prepared with an in vitro-transcribed (IVT) sgRNA versus synthetic cr:tracrRNA. Cells...epitope-tagging experiments. Notably, we could tag >60 transcription factors in a short (3-week) time window; this...epitope tagging experiments: Retrieve Ensembl Transcript ID(s) of your favourite gene(s) from Ensembl .../index.html Design crRNA/ssODN: Using Ensembl Transcript ID(s) as an input query, pick crRNA (guide RNA...strand (strand used by RNA polymerase II for transcription) are more effective for genome-editing events...step by step protocol for the csRNP method is described in our recent article (Dewari et al. 2018), however...

  37. RaPID Detection of RNA-protein Interactions

    Type
    Blog Post
    ...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, Ramanathan...signal (~250-fold enrichment) when compared to a scrambled RNA motif control). BioID2 had no detectable signal...experiments RaPID negative controls include both scrambled RNA motifs and cells transfected with BirA* protein...changes in RC2H1 protein expression and post-transcriptional regulation of SM1v1-containing RNAs could play...IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding proteins...

  38. dTAG - You're it!

    Type
    Blog Post
    ...locus-specific knock-in of FKBP12F36V. Detailed protocols describing the use of these plasmids are available here....example, IZKF1 and IZKF3 are cell-type specific transcription factors with important roles in Multiple Myeloma... (Nabet et al., Erb et al., Huang et al.), transcriptional and epigenomic profiling studies (Nabet et ...degradation, and lead author on the recent paper describing the dTAG technology. Follow him on twitter @behnamnabet... 29581585. This reference contains the full description of the dTAG system. The dTAG system was employed...Featured molecules: dTAG-7 and dTAG-13 Erb et al. Transcription control by the ENL YEATS domain in acute leukemia...

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

    Type
    Blog Post
    ...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery...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...in the genome and directly interacting with transcription machinery.   Well, I’ve got some good news...Hydroxylation, Active DNA Demethylation, Gene Transcription, and Memory Formation. Neuron 79, 6 P1086-1093...

  40. Synthetic promoter AAVs for cell-type specific expression in retinal cells

    Type
    Blog Post
    ...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from..., the Roska lab built synthetic promoters from scratch and tested to see if they drove cell type specific...conserved sequences that are upstream of the transcription initiation sites of at least two genes with ...promoters by repeating mouse cell type specific transcription factor binding sites interspaced with random...designed using stretches of DNA that were transcriptionally active in retinal cell types in mice. ...

  41. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...spreadsheet Data Field Description Plasmid Name Use a descriptive plasmid name and match the... entry process On the Addgene home page, if you scroll over “Deposit” in the mega menu at the top of the... in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options ...does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical application ... Primary Vector Type The vector type should describe how your plasmid is intended to be used. Choose...

  42. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...their coat protein to screen for binding to the target protein. The protocol describes going through two ...protein (sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...enriched pool of sybody candidates are then reverse transcribed into cDNA fragments that are efficiently subcloned...The enriched sybody pools from the phage display screens are subcloned (using FX cloning) into the expression...interest. The Seeger lab used the ELISA technique described in their protocol because it is less susceptible...

  43. Common Injection Routes in Mice

    Type
    Blog Post
    ...off-target pokes to nearby organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume...suspension to create xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind...but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge (bulb-tipped feeding tube...to intermediate (tail vein) Intraperitoneal Scruffing Peritoneal cavity 25–27 < 2–3 mL Intermediate ... < 0.02–0.05 mL Beginner Subcutaneous Back scruffing or mechanical restrainer Back or hind legs; between...Anesthesia Nose n/a < 0.05 mL Beginner Oral gavage Scruffing Throat 18–20 (bulb tipped) up to 10 mL/kg Intermediate...

  44. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    ...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...containing a primer binding site and a reverse transcription template (RTT). The design of the pegRNA has... we performed high-throughput ‘self-targeting’ screens with oligo libraries where pegRNAs are coupled ...cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and building prediction tools...sequence-based prediction models from self-targeting screens such as PRIDICT/PRIDICT2.0 provide valuable insights...your target. Figure 3: High-throughput screening of the effect of chromatin on prime editing efficiency...

  45. Anatomy of a Plasmid Page at Addgene

    Type
    Blog Post
    ...plasmid, how will it be sent to me? Description: This describes how the plasmid or other product (kit...useful to me? Plasmid purpose: This is a brief description of what the plasmid was designed and used for...vector’s plasmid number here. Gene/insert: This describes what has been inserted into the backbone, and ...they’ll end up on the N or C terminus of the transcribed and translated insert. If it is an empty vector... sequencing. Growth in Bacteria: This section describes how to grow the bacterial strain in which the ...

  46. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...resistance gene. These reporters allow you to easily screen or select for cells that are expressing your gene...interest at high levels. Unlike vectors that express screenable or selectable markers from a unique promoter,...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...efficient translation of multiple genes from a single transcript. The two strategies most widely incorporated ...incorporated into plasmids for research purposes are described below. IRES Elements Translation in eukaryotes usually...

  47. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...for transcriptional activation from the 5’ LTR) and Rev (facilitates nuclear export of transcripts). A...Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since lentiviral... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...

  48. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    ...functions such as epigenetic regulation and transcription by using locus-specific chromatin immunoprecipitation...worked extensively with enChIP systems. Can you describe this technology and its advantages? Fujii: In ...biochemical analysis of genome functions such as transcription and epigenetic regulation. To elucidate molecular...enChIP system using CRISPR technology. Can you describe this technology and some of its potential applications... any other technologies that you would like to describe?  Fujii: I believe that "Biology is Method", i.e...Hodaka Fujii lab's Addgene page and click the "Subscribe to Alerts" link in the Addgene Alerts box. ...

  49. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

    Type
    Blog Post
    ...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...promoter in a given cell type. This type of transcription reporting was among the earliest uses of GFP... gene of interest, or expressed from the same transcript as the gene of interest, but after an internal... GFP are available. Others: We’ve really just scratched the surface of the potential applications for ...to identify particular cell populations in drug screens, to visualize micrometastases in nude mice in cancer...

  50. Hot Plasmids - August 2020

    Type
    Blog Post
    ...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...your studies.  Find the plasmids to set up your screening systems at Addgene! Schematic of the evolution...their BioRxiv post, the authors generated and described several improved genetically encoded fluorescent...Takayuki Iwaki, Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion...activating expression of TCF4 while monitoring transcript and protein levels. Plasmids for multiplexed ...
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