Skip to main content
Addgene
Open Menu

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

In Category

We narrowed to 784 results for: SCR;

Showing: 51 - 100 of 784 results

  1. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...start signal sequence (gene start) and a transcription termination/polyadenylation signal sequence (gene...genome which are required for replication and transcription (Figure 1). All viral genes are removed and ...direct minigenome replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The...minigenome is capable of being replicated and transcribed in the cell when the appropriate viral proteins...EBOV polymerase replicates the viral genome and transcribes viral genes. Four viral proteins are required...required for viral genome replication and transcription: NP, VP35, VP30, and L (reviewed in 5). The RNA genome...

  2. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    ... to modulate dCas9 expression, multiple scRNA transcriptional programs can be regulated with dCas9 as ...for CRISPRi are described here: http://www.addgene.org/crispr/qi/ Plasmids described in  Zalatan et ...recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds." Adam has...visualize the concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated...CRISPR-based RNA scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the ...synthetic RNA scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds...domains. Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent target nucleotide sequence...

  3. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...repressor then prevents transcription from occurring, which means that transcription is now OFF.   We’ll...other. For example, the GAL4 transcription factor cannot drive transcription from the QUAS promoter, and...and the QF transcription factor cannot drive transcription from the UAS promoter. This is important because...Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator protein...cannot bind the promoter sequence anymore and transcription is turned OFF. Negative Repressible: A repressor..., but cannot bind the promoter sequence and transcription is ON. Once a co-repressor protein binds the...

  4. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...domain, which activates transcription of downstream genes. Nihongaki et al describe the targeted activation... to activate transcription. Chavez et al. 2015 extended upon these findings by screening for additional... mutagenized and screened for speed and voltage sensitivity. This hierarchical screen led to the generation...and downstream transcription activation or repression. The TCS light sensors described in ACS Synthetic...scientists have been successful in regulating gene transcription using DNA-guided optogenetic tools; however,...recognized by scFV antibodies fused to VP64 transcriptional activation domains. This system enabled the...

  5. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    ... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...induction of a promoter. Below, you’ll find a short description of these three types of inducible promoters, ...removal of the lac repressor (lacI protein) for transcription to be activated. In the presence of lactose ...

  6. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...an alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests...independently of their position relative to a transcription start site. In STARR-seq, randomly sheared genomic...contain an enhancer, it will activate its own transcription from the minimal promoter and its strength can...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced...

  7. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase transcription. This is done...cells. VPR Description VPR fuses a tripartite complex with dCas9 to activate transcription. This complex...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...

  8. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ...Transcription factors (TFs) act as the gate keepers for gene expression, turning transcription on and... regulatory network. The transcription atlas and library The transcription factor library The human... TFs is a useful finding. Transcriptional program changes from this screen successfully differentiated...-scale CRISPR Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi...approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive...differentiation outcomes as well as global transcriptional changes. Using this road map, and what was ...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...

  9. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in...expresses the C-terminal fragment of Cas9 while transcription of the N-terminus is light-dependent (Yu et ...

  10. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...plasmids with the original publication where you described them. Search for your article as you would search...published. Plasmids can be linked to a peer-reviewed manuscript OR a preprint. In the case of a preprint submission...to update the publication information once the manuscript is published in a peer-reviewed journal. To start...simply enter your preprint title or a general description of the deposit. Select your desired distribution...distribute, or Addgene finds your journal publication describing the plasmids, or (for deposits submitted after...selected your publication or entered your deposit description, the next page will allow you to add plasmids... article. Note that if you entered a deposit description, this will become your article title.   ...

  11. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to reverse transcribe back into ...they copy themselves as RNA transcripts and then use their reverse transcriptase (RT) enzyme to convert back...mutagenesis screens that detect loss-of-function or gain-of-function mutations. In these screens, the transposons...transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters and enhancers...protein is either a transposase or a reverse transcriptase, depending on the mechanism of transposition...pol. The pol polyprotein encodes the reverse transcriptase and integrase enzymes which are required for...poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    Figure 1: Overview of...

  12. Bioinformatics at Addgene

    Type
    Blog Post
    ...copying and pasting pieces from our existing script into the script block of the Nextflow process.  Next, we...of data. In this two-part blog series, we will describe the importance of bioinformatics at Addgene and...possible solutions for each in the form of one-off scripts or programs, our goal was to find a suitable software...sequential computational tasks for which existing script(s) have been developed for a singular purpose (...sequence assembly), but the order and usage of the scripts needs to be customized in the pipeline for the ...In Nextflow, a process defines which command or script will run and can be thought of as a particular ...Prior to using Nextflow, we routinely wrote Python scripts, queried our database with SQL or Django, and executed...

  13. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    ...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using ...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...producing a wall of results that can only be described as bigly. But such a project should not be undertaken...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are... in two sets, each with 4 gRNAs per kinase, so screens can be performed with either 4 or 8 gRNAs per gene...found in genome-wide libraries, and thus can be screened in pooled format at a more-typical scale of tissue.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...

  14. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...need to screen hundreds of clones looking for your edit of interest. For all of the screening strategies...phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base...needle in a haystack before going on to generate and screen individual clones. If a clonal line isn’t your ... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ... the minimum number of clones you would have to screen to identify a knockout clone. For example, if your...interest. Thus, to find a null clone, you should screen, at the very least, 4 clones. Did you use a dual...

  15. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...ncRNA locus (Ho et al. 2012). For large-scale screening, sgRNA libraries are useful for targeting thousands...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...the removal of exons. Disrupting promoter or transcription start site function would also help to reveal...

  16. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening - An excellent protocol paper by...Feng Zhang lab describing construction, lentiviral production, transduction, screening, and validation...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc. 2017 Apr;12(4):828-...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...the library sample, and any other information or scripts provided by the depositing lab for data analysis...

  17. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...activation of transcription in bacteria by co-opting σ54-based transcription. Transcription from σ54 promoters... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...been created in lentiviral systems for mammalian screens, which enables researchers to maximize the chance...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

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

    Type
    Blog Post
    ...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...silence the target gene(s) either transcriptionally or post-transcriptionally. In plants, different dicer-like...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ...alternative start codons, and/or the phenomenon of transcriptional adaptation (Smits et al., 2019; Sztal and Stainier...RNAi in the context of a high throughput survival screen found that either technology was equivalently precise... researcher, the traditional hpRNA and visual screening method was limited in two substantial ways: variable...

  19. Hot Plasmids - February 2022

    Type
    Blog Post
    ...expanded the genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability ...genetic screens, and this optimized toolkit furthers Cas12a’s potential in future genetic screens.   DeWeirdt...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...Cas12a has generally been underutilized in genetic screens because of its lowered editing rate. This research...aimed to increase the potential of Cas12a genetic screens by optimizing enzyme activity using an enhanced...

  20. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    ...estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well ... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...Tyler J. Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the...deposited plasmids encoding synthetic yeast transcription factors that can independently control expression...expression of your genes of interest. These transcription factors consist of the GAL4 or ZIF268 DNA binding...domains and the MSN2 transactivation domain. Transcription factor activation via the appropriate ligand...addition to plasmids encoding these synthetic transcription factors, the El-Samad lab has deposited plasmids...

  21. All in a Twist: dsRNA

    Type
    Blog Post
    ...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...will dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly...thought to be associated with bidirectional transcription. In an uninfected cell, dsRNA rarely circulates...short, dsRNA molecules designed to degrade mRNA transcripts. RNAi activates the RNA-induced silencing complexes...elements can generate dsRNA by bidirectional transcription or as inverted repeats. These elements are almost... produce dsRNA as a result of bidirectional transcription of their circularized DNA. These dsRNAs are ...structure produced is known as an R-loop. During transcription, RNA is synthesized from a DNA template, inherently...

  22. Plasmids 101: Modular Cloning

    Type
    Blog Post
    ...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...specifically refers to the organizational schema described above, and not any particular cloning technique...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. ... with DNA, and these complexes regulate the transcription of specific genes. What if one of those genes...

  23. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    ...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...potential options and cloning methods, but please scroll down for plasmids suitable for other model systems...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter... Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease...like AAV. In addition to the mammalian option described below, plasmids for making polycistronic gRNAs...

  24. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...TALEN-based LITE system, which uses light to regulate transcription, also includes light-regulated histone methyltransferases...check out our CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused...enhancer regions. These marks are associated with transcriptional activation. Created with BioRender.com. ...Tet1-CD to decrease methylation and activate transcription. A lentiviral vector with the same modifier,... demethylation is associated with increased transcription. Created with BioRender.com. Tet1 initiates...

  25. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use...the best uses for Cas9 activators is in genetic screening. gRNAs targeting every gene in the human genome...interactions. Additionally, large scale CRISPR-based screens can be used to gain insight into protein structure...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides by...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568:511–516 . https://doi.org/10.1038/s41586...

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

    Type
    Blog Post
    ... 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...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... in all kinds of different assays, not just transcription factors. The second vector is a homology directed...

  27. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...proteins involved in the activation of transcription including transcription factors, phage proteins, and RNAP...citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria As RNA interference...targeted to a promoter or gene body can repress transcription by physically blocking the elongation complex...complex from binding the DNA or extending the transcript. Different Cas orthologues and subtypes function ...regulatable CRISPR system effectively silenced transcription of one or more genes in E. coli. and in a mammalian...reliably knockdown expression of multiple target transcripts simultaneously. Marraffini lab's CRISPRi/a plasmids...

  28. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...tools. Scroll down to see a table summarizing all of them! PE2: Improving reverse transcriptase efficiency...consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase is guided to the DNA target...hybridizes to the target site, and the reverse transcriptase domain copies the desired edit, directly polymerizing...Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase to the C-terminus of Cas9 H840A nickase (PE1...efficiency Building upon prior reverse transcriptase research, the Liu lab created and evaluated 19 PE1 variants...inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch ...PE2-PE5 strategies. PEmax contains a reverse transcriptase with a sequence optimized for human codons; ...

  29. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...and the amount of time spent shopping a manuscript. Manuscript writing can take weeks to a couple of months...for and how can you do it well? Below, I briefly describe some tips for a successful postdoc fellowship..... Now work backwards. To publish a submitted manuscript in a high-profile journal typically takes 6-12...for a Group Leader position, key first author manuscripts need to be ready to write by Year 3.5. Thus, ...end goal of having sufficient data to write a manuscript. Consider questions like: What figures are missing...model?  PFs need to be regularly thinking about manuscripts. The other items will follow. If you get more...validated? How will the results fit into a future manuscript? You could easily spend a day or a week planning...

  30. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...CRISPR screens By Emily Bentley Building on their CROPseq method for optical pooled CRISPR screening, Paul...Multiplexed perturbation and decoding in pooled CRISPR screens Improved prime editor PE7 Antibodies to facilitate...multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi (Walton et al. 2024). This...enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors for CROPseq-multi... with tRNAs, which are cleaved out of the RNA transcript by endogenous processing enzymes (Figure 1). ...processing only occurs on the RNA polymerase III transcript, the sgRNAs were encoded on the lentiviral minus...multiplexed perturbation and decoding in pooled CRISPR screens. bioRxiv 2024.03.17.585235; doi: https://doi.org...

  31. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...engineer specific DNA edits and to perform functional screens to identify genes involved in a phenotype of interest...the need for single cell cloning and subsequent screening for successful edits. This is a very time consuming... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...examined whenever possible when using knockout or transcriptional modulation approaches. Avoiding off-target ...Further, large-scale datasets of hundreds of genetic screens using genome-wide libraries have shown high concordance...

  32. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...during your in vivo work is to screen you gRNA in vitro first. In vitro screening is a simple and fast method...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...Several companies supply gRNA sequences, such as GenScript, ThermoFisher Scientific and OriGene, or you can...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...gRNAs to Target Mouse Genes To begin the in vitro screening process, you should first develop primers to the...embryos to develop. Once they are born, it’s time to screen them for your desired edit. Good news! Remember...no mutation, costing money and space. As a rule, screen all of your pups unless you have an enormous litter...

  33. Clear and Accessible Writing

    Type
    Blog Post
    ...the use of alt text to describe images in online spaces. Alt text is used by screen readers to provide visually...Students Writing Scientific Manuscripts Tips for Improving Your Next Manuscript...example. Remember, our goal is to use words to describe the image in a way that helps visually impaired... your alt text, use all of this information to describe the image helpfully. You and your readers are ...learned a lot from listening to others' alt text descriptions, and we’d love to hear yours as well.  More ... You can use them to help you write an entire manuscript, or you could use them to write alt text for ...for any image you’re struggling to describe in the results. And many journals accept alt text, so you can...

  34. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    ... to create a complete transcription factor, activating luciferase transcription. Tang et al. coined this...Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence...Using nanobodies to create GFP scaffolds for transcriptional activation The project began with a frustration...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...this system T-DDOG (Transcriptional Devices Dependent On GFP). In testing the specificity of their system... dsRed, mCherry, and TdTomato do not induce transcription. Thus, T-DDOGs can be used in combination with...T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, and Tang and...

  35. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...specific markers. The Screening Criteria While choosing which antibodies to screen, we kept in mind our...We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed a criterion...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...multiple people in the lab conduct “independent screening”, where they look at the same section and determine...reproducibility, each antibody is independently screened twice, confirming it will produce the same results...available 2D/3D mouse atlas from antibodies that we screen. This atlas will enable scientists to have an intuitive...

  36. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ... then be fused to effector domains such as transcriptional activators or repressors. These fusion proteins...Consequently, fewer genetic tools for targeted transcriptional and epigenetic regulation have been validated...Furthermore, among those Cas9 orthologs that have been scrutinized, the majority have been shown to exhibit more...inexpensively generate a potent Cas9-based transcriptional regulator without making any functional changes...contains an RNA adapter capable of recruiting a transcriptional activator, robust regulation of gene expression...incorporated into the truncated gRNA recruits a transcriptional activator to the Cas9-gRNA complex. When this...complex is directed to a target upstream of the transcriptional start site (TSS) of a gene of interest, it ...

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

    Type
    Blog Post
    ... ready for experimentation. In 2014, several manuscripts published in Genetics detailed approaches in ...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal ...parental animal, are rescued progeny, which makes screening extremely rapid. This stringent selection allowed...

  38. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...large scale functional screens. For more information on these new CRISPR screening tools, see our CRISPR... Lentiviral CRISPR libraries for knockout screening New systems have been developed and deposited ...CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries expand upon ...CRISPR Libraries Enable Genome-Scale, Knockout Screening. Also, check out our updated CRISPR-Cas resources...minimizing confounding secondary effects? An approach described as “knocksideways” (a British idiom for “taking...Chrimson Through de novo sequencing of 127 algal transcriptomes, as well as further optimization through engineering...two-color optical excitation of neurons. The paper describes five previously unknown channelrhodopsins from...

  39. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    ...isolated and screened. Depending on the marker, traps can be used as high-throughput screens to introduce...Each trap uses a slightly different approach (described in detail below), but fundamentally the reporter...models. Why traps are useful Traditional genetic screens employ various on/off switches to measure the function...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems ...integrate within an exon of a gene to generate a transcriptional fusion. This may disrupt the endogenous function...spliced into the gene product resulting in a transcriptional fusion, much like promoter traps. While this...likely disrupt the gene it monitors due to transcriptional fusion.   Examples of promoter, enhancer...

  40. Single-cell tracking of lineage and identity with CellTag

    Type
    Blog Post
    ...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...cell technologies, such as single cell RNA-seq (scRNA-seq), now allows scientists to identify the gene...cells that successfully reprogram. Unfortunately scRNA-seq only captures the gene expression of cells at...identifiers act like a timestamp and help demultiplex the scRNA-seq data and build multi-generational cell lineage...reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like...reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), ...three days, a sample of cells was collected for scRNA-seq analysis and the remaining cells were maintained...

  41. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    ...Detect Zika Virus The RNA-based toehold switches described in this post are engineered to bind RNA sequences...Boston University School of Medicine, this post describes a tool you can use to study basic virus biology...sequences required for viral replication and transcription as well as reporter genes. To learn how you ...researcher in the Silver Lab at Harvard University, describes R-bodies, “force generating protein machines that... Connie Cepko’s Lab at Harvard Medical School describes how AAVs function and indicates important things...CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that can be used to quickly...barcode small libraries of CRISPR gRNAs to use for screening experiments. Get the full details on how to make...

  42. How to Keep a Lab Notebook for Bioinformatic Analyses

    Type
    Blog Post
    ...commonly used scripts: If you edit a general purpose script for a single use, save the script as originalName_descriptionOfEdit...be in the scripts that you run. Rather, this is where you outline your logic and the scripts and input...immediately write a text document with a brief description of the directory contents, at a minimum. This...for all of your analyses. The same is true for scripts. Naming conventions Give every experiment a number... remains in tact! Take notes Comment in your scripts: Comments on what a given line of code does are...

  43. A Look at Addgene's QC Process

    Type
    Blog Post
    ...effort into our quality control (QC) processes to screen for and reduce these errors. These processes allow...types of data for each of their plasmids, from descriptions of key components of the plasmids to partial...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ...will contact the depositing lab to review the discrepancies. Often, the identified issues will not affect...

  44. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    ...with a publication in Science describing the development of “transcriptors,” transistor-like digital genetic...cellular computing. These logic gates built from transcriptors are available at Addgene as a BIOFAB kit comprised...comprised of well-characterized bacterial transcription and translation initiation elements. Open science...with the central dogma - replication of DNA, transcription of DNA to make RNA, translation of RNA to make... and observed that we humans find it useful to describe and interact with the world using words like OR...The future of synthetic biology Addgene: You've described the pieces in these kits as the beginnings of ...systems today who are building 10-gene systems from scratch. (They may be building genomes, but that's just...

  45. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ...genome-wide screens, introducing bias into all work based on these and any other CRISPR screens using the..., race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic...targets and related biomarkers . Data from such screens are often uploaded to the Cancer Dependency Map...Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify the ancestry... American, Africa, or South Asian) used in the screens based on the cells’ germline genetic variations...about 90% of the cell lines used in DepMap CRISPR screens are of either European or East Asian ancestry, ...actually reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...

  46. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...dCas9 has focused on transcriptional activation using a fusion to a transcriptional activator such as VP64...repression of transcription through binding a promoter region to inhibit association of transcriptional activators...region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific genomic region using...locus of interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an ...plasmid. Experimental use Since the initial report describing CRISPR-mediated purification of a specific genomic...Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes." Cell 154.2 (2013): 442-451. ...

  47. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...backbone. This type of primer pair is also great for screening clones created with the same backbone but that...is, allowing you to use the same primer pair to screen for the presence of many different inserts. The...test your colony PCR primers before using them to screen colonies. The best way to do this is by using your...insert and if it's in the plasmid Can be used to screen clones that were created with the same backbone... background, the more colonies you will need to screen.   Saving clones for later culture: At this point...expected results for the three primers previously described. When using insert-specific primers (1), positive...

  48. Modulate the Activity of 17 Signaling Pathways with One Kit!

    Type
    Blog Post
    ...a plasmid kit designed to do exactly that. As described in the journal Science Signaling in December 2014... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ...

  49. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    ...targeting dCas9 to transcriptional start sites was sufficient to repress transcription by blocking its initiation...desired edit adjoined to the sgRNA, a reverse transcriptase, and a nCas9. While this system has been shown...Prime editor engaging target DNA. The reverse transcriptase polymerizes DNA onto the target strand based...been exploited to localize factors such as transcriptional activators and repressors to target genes. ...repression requires targeting dCas9 along with transcriptional repressors to the promoter region of the gene...associated box (KRAB) is a naturally occurring transcriptional repressor domain which is frequently fused ...activators consists of dCas9 fused to a single transcriptional activation domain - VP64.  Later generations...

  50. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...assays for your screen result may or may not actually help. Ultimately, if you cannot describe the likely ...of experiments or a laundry list of molecules to screen has unclear utility. You cannot predict what will...cannot either. Therefore, you need to be able to describe experimental approaches in terms of the question...approach, anticipated outcomes, and preferably a description of the payoff if the aim is successful. Note ...the aims and clarify what type of aim you are describing. It's easy to be overly ambitious – a common ...distinguish mechanistic questions from open ended screens and data collection. Organize your research vision...accomplished in 5-10 years? You need to be able to describe what success looks like. If you are successful...
Showing: 51 - 100 of 784 results