...Tet-controlled transactivator (tTA, also expressed by your transfer vector) binds to the TRE and activates the expression...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...improving the safety of these vectors is still an active area of research due to the possibility that mutation...have to be aware that there is often  background activity and it requires continuous administration of Dox...GFP encoded virus, and the reverse transcriptase activity assay uses qPCR to quantify viral RNA. All of ...microglia. This property makes the lentivirus an attractive choice for a gene transfer vector. Lentiviral...are working with proto-oncogenes or with human samples that have not been tested for HIV.  If not using...

...or is engineered not to cleave DNA and instead activates/represses gene expression by various mechanisms...nicking/cleaving of DNA occurs, not for deadCas activator/repressor experiments where only localization ...by Cas does not always correlate with catalytic activity at those sites, especially when DNA mismatches...for non-cutters If your Cas experiment involves activation/repression or any other effect which does not...RNA-seq would be an appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would...If you are performing CRISPR library screens, the same principle holds true - the higher the number of ...

...editing of genomes. It will also allow simultaneous activation and/or repression of multiple genes, and more...Editing CRISPR-Cas9: Tips for Optimizing sgRNA Activity Lentiviral CRISPR Libraries Enable Genome-Scale...to modify the expression of multiple genes at the same time.” For more details on how to apply the new ...

...you are trying activate a gene, you should design a control that shows maximal activation. Likewise if ...cells. Regardless, the amount of reporter protein activity correlates to both the amount of DNA transfected...were derived from the same source and were incubated on the same plate at the same time. Although the replicate...separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The replicate...provide the baseline/standard against which other samples can be compared. The Empty Vector Control (mentioned...control in this experiment could be an shRNA—in the same backbone as Plasmid A—that does not target any mammalian...". Biological replicates are what makes up your sample size (aka your n value)" and should come from multiple...

... be used to knockout, activate or repress target genes in plants using the same general experimental design... al., 2013). Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB...KRAB or dCas9-SRDX) can also be used to activate or repress target genes in plant cells, respectively. ...carries Cas9-containing plasmids for knockout, activation and repression of target genes in plants and ... expression of both Cas9 and the gRNA off of the same plasmid. Delivering CRISPR components to plant cells...

... hyperthermostable Cas9 CRISPR nuclease that is active up to 100°C.  The IgnaviCas9 protein is 1,241 amino...time periods and workflows where CRISPR nuclease activity is desired and time or protocol steps can be saved...highly complex hot spring sample. The sample was separated into smaller samples of 5-10 cells each for sequencing...biological dark matter and so the lab organized a sampling trip to Yellowstone National Park (YNP). Extremophiles...from these extreme environments. Gearing up for sample collection at the hot springs My husband, Paul ...post doc in the Quake Lab, volunteered to do the sampling and I happily agreed to accompany him to YNP the... and two deep cycle batteries for power so the samples could be kept frozen after collection. In September...

...signal. The same effect can be achieved for a fluorophore that relies on dimerization for activation, only ...Using a combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with...Experiments Learn more about GFP Controlling Protein Activity with GFP Resources at Addgene.org Find the Spinach...context of a paired fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their deep... Jaffrey lab asked:  Could an RNA aptamer do the same thing? Using the HBI derivative 3,5-difluoro-4-hydroxybenzylidene...· February 2008. 11. Svensen, Nina and Jaffrey, Samie R. “Fluorescent RNA Aptamers as a Tool to Study ...

...composed of two parts: 1) an activatable protein (the FAP - fluorogen activating protein - dL5** in this case...), and 2) a fluorogen (the TAP - targeted and activated photosensitizer - MG-2I in this case). The fluorogen... blood circulation of following FAP-TAP light activation. Video: https://www.youtube.com/watch?v=xOtH5bs4HiI... to produce reactive oxygen species (ROS). The ROS produced by photosensitizers inactivate target proteins...exposure times (5 minutes vs 10 seconds) to achieve the same level of killing as Mg-2I-dL5**. Mg-2I-dL5** also...allows for precise light-based control of protein inactivation/cell ablation while having limited collateral... Hahn, K. (2008). Chromophore-assisted laser inactivation in cell biology. Trends in Cell Biology,18(9...

...of interest (analyte), and accumulation of ATP activated GFP expression (receptor), then the transducer... muscle contraction, and calcium channels are activated for neuronal action potentials; it’s an ion of...cellular dynamics during so-called housekeeping activities.       Viruses and biosensors So how...monitoring of different factors, in different or the same cellular location, all at once! Small molecules ...

...regulatory regions that are necessary for myosin activity and thus interaction with actin in nonmuscle systems...restricted to the dorsal mesoderm, is necessary to activate bagpipe which is localized to segmental clusters...Pascale, and Roger Karess. "Myosin light chain–activating phosphorylation sites are required for oogenesis...Jianqiao, and Helen White-Cooper. "Transcriptional activation in Drosophila spermatogenesis involves the mutually...call a gene required for spermatogenesis after a Sesame Street character? The scientists who discovered...

... and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 .... Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the editing efficiency...system to identify and enrich for base-editing activity The reporters mentioned above all rely on GFP ...reporter system to identify and enrich base editing activity. Nucleic Acids Research 48:2841–2852 . https:/...by canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing DNA strain which...to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202, eGFP L138, and eGFP...

...Amplification Protocol (DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540...Re-Amplification Protocol (PDF, 71 KB) CRISPR Activation Human Subpooled CRISPRa-v2 Libraries 12,285...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab,...library from Addgene, they’ll need to amplify the sample for further downstream applications. As individual... all of the bacteria can grow and compete in the same container, making it easier for a smaller, recombined...

...within the target gene. CRISPR can also be used to activate or repress target genes without permanently modifying...CRISPR libraries have been created to knock out, activate or repress target genes by combining a gRNA library...Addgene carries CRISPR libraries for gene knockout, activation, repression, and barcoding.  3) Are you trying...Enrichment or depletion of multiple gRNAs targeting the same gene can be strong evidence that a particular gene...

...deposited PSAM AAV, which encode engineered ligand-gated ion channels that can be activated with FDA-approved...expanded our inventory of chemogenetic AAV for activating and inhibiting neurons. Minimally-tagged DREADD...size 20 µL samples of control AAV that scientists can use for serotype testing. These samples are available...serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic inhibition Flp expression ...ability to detect the neurotransmitters and other bioactive molecules is integral to the study of neuroscience...Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are...ppAAV-CKIIa-stGtACR1-FusionRed New tetracycline transactivators Plasmid Serotype Name 99120  AAV1 pAAV-ihSyn1...

...original method, we used the Tetracycline trans-activator system to boost the expression levels, and we ...we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1)....not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1...time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks...excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this procedure..."Optical clearing and index matching of tissue samples for High-resolution fluorescence imaging using ...

...Similarly 90% of active research scientists believe climate change is mostly due to human activity while much...opinion do not seem to take note. For example, 91% of active research scientists (scientists who hold a doctorate...conversation about science’s role in government as well as active advocacy will be required. Information on how ...politician Rush Holt said, government must have the same fundamental attribute that enables science: “humility...

... scared to engage with them. Comment on their activities and participate in discussions. Get your voice...Twitter to promote your other science communication activities, e.g. tweet it when you publish a new blog post...step I have taken in my career. The moment, I got active with it, new doors started opening up. My current...requires lots of data. Get your community involved in sampling or bioinformatics analysis or upload your data...

...cut-to-mutation distance"  Even though we had controlled over-active Cas9 and repeated our experiments with blocking...have sufficient on-target and very low off-target activity, but should also mediate a DSB at optimal distance... the desired zygosity. Furthermore, re-editing activity of the guide RNA should be minimized by incorporating... events were corrupted by unwanted indels on the same allele, probably due to concomitant NHEJ repair....blocking mutation and our intended mutation were on the same oligo, the intended mutation rarely got in. It seemed...mixed with another ssODN template that contains the same CRISPR/Cas-blocking mutation but not the intended...

...ways to incorporate science themes into our fun activities and Halloween is no exception. For the past 10...grasshoppers, including those linked to neurotransmitter activity and geotactic behavior (Biron et al., 2005).   ... 2015). There, it divides into two cells. At the same time, it sucks out its contents (hence the name ...

...Reproducibility for Everyone Workshop included this interactive activity during their session. Image from Reproducibility... more initiative. “You have to actively reach out. It’s more proactive versus organic networking,” she...both days. pic.twitter.com/4MysvpzwkP — Samuel Guay (@SamGuay_) June 18, 2020 Final thoughts on virtual...at once, virtual conferences may have apps or interactive calendars that you can use to help plan your ...the Q&A in the chat box. Presenting? Make it interactive! As a presenter, it can be difficult to keep ...spend with the conference. “If you want to have the same experience as an on-site conference, you really ...