...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase...VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using a single copy of VP64 per each...expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to ... Signal Amplification in Gene Expression and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/...

...in this post: Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl...New ready-to-use viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz...SARS-CoV-2), the virus may acquire mutations to escape these therapeutics. Jesse Bloom’s lab at the Fred...From Starr et al Cell., 2020. (B) Mapping antibody-escape mutations with the yeast display library. From ...characterized them by ELISA, homogeneous time resolved fluorescence, and suspension bead assay (Figure 2). After...

...co-express your gene along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow...incorporated into plasmids for research purposes are described below. IRES Elements Translation in eukaryotes...co-express your gene of interest along with a fluorescent protein or selectable marker, it is easiest to...genes at equivalent levels upstream of an IRES fluorescent reporter for easy detection.5  Just released:...efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim...

... FAST system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors...of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa) (Zhou et al., 2018). pdDronpa... flanking the DNA-binding cleft of Cas9, the fluorescent domains dimerize and prevent DNA binding in the...chromatin architecture (Hoffman et al., 2020). Fluorescent dCas9 proteins can be used to identify specific...gRNA and a dCas9 fusion protein with three red fluorescent tags in U2OS cells. Upon light activation, dCas9...figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated that the...

...until new inflorescences begin to grow. This will also increase the number of inflorescences overall, ...and C terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or affinity tags (...transformation, you can cut off the primary inflorescence (the flowering portion of the plant) near the.../books/about/Arabidopsis.html?id=IfZAMNPWVk4C&redir_esc=y (Accessed: 21 September 2018). Additional resources...

...Rogers, a postdoctoral researcher at the Friedrich Miescher Lab of the Max Planck Society. Zebrafish (Danio...tissues such as blood cells or neurons can be fluorescently labeled, allowing researchers to observe changes...guest blogger Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society. Katherine W. Rogers...researcher in Patrick Müller’s group at the FriedrichMiescher Laboratory of the Max Planck Society in Tübingen...about zebrafish behavioral studies Download the fluorescent proteins 101 eBook Read our plasmids 101 blog...

...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-...High-throughput screening strategies Although the tools described above make CRISPRa more accessible to neuroscientists...The pooled nature of this experiment requires fluorescence activated cell sorting (FACS) to isolate differentiated... in vitro and in vivo applications, the tools described above make CRISPRa in the brain relatively easy...

...sample. A variety of chromogenic, chemifluorescent, and chemiluminescent substrates are available. Labs ...standard plate reader as opposed to chemifluorescent, and chemiluminescent methods that require specialized...peroxidase (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we ...

...successfully and expressing the protein. For example, fluorescence microscopy images resulting from our experiment...was not successful due to the absence of GFP fluorescence from the living, viable cells. This result presents...positive control, the internal control vector, was described earlier. Once you are sure your conditions are...produces the expected effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid...come from multiple, independent sources. We'll describe these a bit more below, but the papers cited in...

... Biology Reference Introduction This protocol describes how to use droplet digital PCR (ddPCR) to titer...

... Biology Reference Introduction This protocol describes how to extract RNA from tissues or cells without...

...several secondary antibodies, increasing the fluorescence signal intensity. However, you need to be careful...sorting based on a single (lineage) marker, while fluorescence-activated cell sorting (FACS) enables you to...off by enzymes, turning the resulting molecule fluorescent and membrane-impermeant. With each cell division...(Figure 4a), yielding a stepwise decrease in fluorescence intensity in a (histogram) flow plot (Figure...division half of the dye molecules are lost and the fluorescence signal weakens. (b) The heterogenicity of proliferative...

...site-specific incorporation of phosphoserine (as described below) provides researchers with a valuable tool...context-specific effects of UAG suppression, as described (6). To determine the stoichiometry of phospho-incorporated...phospho:non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid...-Sung, et al. "Expanding the genetic code of Escherichia coli with phosphoserine." Science 333.6046 (2011...al. "A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation...

...of working with fruit flies is their ability to escape their habits and live in the lab itself. Many of...Light Interval System, or FLIS, incorporates a bioluminescence protein into the chitin that can be activated...everywhere find and re-contain any specimens that escape during routine lab activities.  Here, we demonstrate...plasmid-based system for locating and containing escaped flies. Nature 4:1-22. https://doi.org/4.01.2022...

...process of mouse model generation simpler, as described in an earlier post on generating CRISPR mouse ... make. For example, large insertions, like a fluorescent protein knock-in, suffer from low efficiency ...with your desired insertion feature (e.g., a fluorescent protein), you can amplify the desired region ...most of the commonly used cassettes, such as fluorescent proteins (EGFP, mCherry etc.), recombinases (...'d use ivTRT to create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids...

...delivering shRNA, CRISPR/Cas9 components, and fluorescent sensors. Retroviral vector background Until ...lentivirus-derived vectors are capable of transducing certain quiescent or terminally differentiated cells such as macrophages...interest is expressed together with a marker (Fluorescent protein or selection marker) allowing for selection...process is to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the...quantify viral RNA. All of these methods have been described in a previous Addgene blog Once you know your ...

...Specific C-to-U Exchange, or RESCUE. Read the paper in Science Find RESCUE plasmids If you have a new... toolkit for targeted genomic integration of fluorescent tags in neurons. Their toolkit named ORANGE (...Editing) mediates targeted genomic integration of fluorescent tags in neurons.  Read the paper in bioRxiv ...

...camelids too, though not just for the reasons the UN describes. In honor of the Camelid family, I wanted to take...of times on this blog over the years and have described how they compare to other antibodies and affinity...single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens...sdAbs in diagnostic applications. For example, fluorescently and radioactively labeled sdAbs have become ...

...database describing nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all ...authors of the paper in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by...Cellosaurus received a report that HT-55, which was described as a colon cancer cell line, is actually a rectal...

...(FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...via applications such as western blot and immunofluorescence. GST  Glutathione S-Transferase (GST) is ...