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... cultured cells as well as more difficult conditions such as model systems and primary cells. References...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... human CRISPR libraries. Brunello: human CRISPRko sgRNA library The Brunello library consists of 77,441...criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled library...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion... viability screens. Based on these metrics the Brunello library was more effective than other popular ...genes. In the subsampling analysis of the sgRNAs, Brunello even with only a single sgRNA outperformed another...

...measure, and control molecular signals, cells, and groups of cells in order to understand their activity...Fluorescence-activated cell sorting (FACS): This is a type of flow cytometry that separates mixtures of cells into distinct...Thus, FACS can be used to separate cells expressing GFP from cells that are not. Developmental/transgenic...(GFP) has likely existed in creatures such as jellyfish for millions of years; however, it took until ... localization profiles, acting as a biosensor or cell marker, studying protein-protein interactions, visualizing...consisting of 238 amino acids derived from the crystal jellyfish Aequorea victoria. It has a fluorescent emission... was non-toxic and could be expressed in living cells, enabling the study of dynamic, physiological processes...

...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...pol, and env), the isoforms of these proteins, as well as the viral long terminal repeats (LTRs) are different...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...

... Oct 7, 2020 by Gabrielle Clouse. This post was contributed by guest bloggers Marcelle Tuttle and Alex...differentiate induced pluripotent stem cells (Chavez et al., 2015) into neuronal cells by activating genes that cause...drug targets by finding genes that cause cell death in cancer cell lines upon activation (Gilbert et al.,...reasonably well.  Best of luck on your experiments!  Many thanks to our guest bloggers Marcelle Tuttle ...Repression and Activation. Cell 159:647–661 . https://doi.org/10.1016/j.cell.2014.09.029 Konermann S, Brigham...and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/10.1016/j.cell.2014.09.039 Wong N, Liu W,...activation allows for exploratory-based research as well. For example, CRISPRa can be used to find potential...

... origin as well as a selectable marker and a desired genetic payload (ex: GFP). Thus, cells can be both...bee gut microbiome species such as Snodgrassella alvi and Gilliamella apicoli. The plasmids could also ...strategy for bacteria to count cell divisions. The technique, called distributed cell division counting (DCDC...one bright particle that segregates into one cell during cell division. To achieve the agglomeration of ...promoter. Cells are first induced with arabinose to begin expressing the SAP-RFP fusion. Then, the cells are... new particles. With subsequent cell divisions, the fraction of cells containing the particle exponentially...plasmids to both Gram-positive and Gram-negative cells.  The Wang lab further developed a modular suite...

...homodimerize in cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays... quantified for individual cells, and the number of 'normal' looking cells can be counted. This assay ...living cells. Strikingly, the OSER assay reveals that tagRFP does not act as a true monomer in cells (Constantini...a non-aggregating fluorescent protein. In the jellyfish Aequorea victoria, AvGFP forms a homodimer. In...tolerated in some applications (e.g. labeling of cells or tracking promotor activity), but it is problematic... affinity and has been used to demonstrate that yellow fluorescent protein homodimerization is characterized...fluorescent proteins interact or oligomerize in cells? This question is largely unanswered. Ideally, fluorescent...

...successfully infected/expressing cells by flow cytometry. The isolated cells were injected into mice and imaged...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...acceptor. NLuc is an excellent BRET donor due to its bright luminescence. Intracellular Calcium Sensor Yang...to changes in intracellular calcium and is useful for direct imaging of events in cells and brain explants...could “clone by phone”. One of my professors was excellent at phone cloning with labs around the world and...luciferase by Promega’s Advanced Technologies group as well as evolution of the coelenterzine substrate to the...

...active wavelengths to reach zooxanthellae, the coral’s symbiotic dinoflagellates that are photosynthetic ... them into your cells, you can simply look for blue colonies to determine which cells have taken up the... that the cellular fitness cost varied greatly between chromoproteins. In some cases, cells containing... mScarlet as the donor in HeLa cells.   Find ShadowR for mammalian cell expression here   Now that you... is full of striking visuals from fluorescent jellyfish, bioluminescent phytoplankton, and colorful corals...described an unusual blue-colored protein from the jellyfish Cassiopea xamachana (Blanquet and Phelan, 1987...cloning experiment worked just by looking at the cells on the plate. For example, if your plasmid contains...

...precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV lysis buffer and freeze...packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids...preparing reagents, I choose to only harvest the cell pellet. My yield would be higher if I used PEG precipitation... my experiments, I get enough virus from the cell pellet alone. If yield is important, or if the viral...bath to release the virus particles from the cells. Cell lysates are then treated with benzonase immediately... genome that's then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses...five days after transfection, the AAV-containing cells and media are harvested and purified. In total, ...

...that cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted expression...this plasmid pool are injected into an animal with cell-type-specific Cre, the polyA sequence is inverted... library to infect an animal expressing Cre in a cell type of interest, a scientist can isolate DNA from...(s) of the capsid gene successfully infected the cell type of interest. Repeat rounds of infection and...capsid variants with increased ability to infect the cell type of interest, which, in the case of Chan et ...it was actually engineered to target the CNS. How well do these AAVs transduce the CNS and PNS? Both plasmids...

...efficient gene deletion in B cells, T cells, macrophages, and dendritic cells, with no deleterious effects...to HEK293FT and HeLa cell lines. When the miRNA activity profile for a given cell type is already known...or ZFN to edit immune cells poses challenges such as the inhibition of immune cell differentiation. Furthermore...immune cell activity. However, a CRISPR-based tool for in vivo functional analysis of immune cells has been...construct in bone marrow stem cells from Cas9-expressing mice, and used the cells to reconstitute the immune...vivo using ultrasound in Escherichia coli and Salmonella Typhimurium. This work demonstrates engineered...post-transcriptional gene expression across the majority of cellular pathways. Despite several years of research, the...

...fluorescence activated cell sorting (FACS) to isolate differentiated from undifferentiated cells at the end of ...these cell types are used. The lack of good flow cytometric antibodies for many CNS-resident cell types...for which CRISPRa is well-suited, including studying mRNA dosing effects, cellular reprogramming, and disease...that drive neuronal fate and reprogramming." Cell stem cell 23.5 (2018): 758-771. PubMed PMID: 30318302...-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at University of Rochester Medical Center. During...relationships in defined substructures of the CNS as well as individual neuronal populations.  The Yang Lab...lentiviral vectors for in vitro and in vivo CRISPRa (Savell et al., 2019). This dual lentivirus system pairs...

...specific cell types (populations) within neuronal networks in living animals. To accomplish such cell specific...individual cells. With this technique, a Neuroscientist can see the full dendritic arbor of a single cell with...Cre-mediated possibilities for site specific (and often cell type specific) control of DNA recombination and ...expression can be advanced by the coordinated use of fellow SSR system FLP-FRT. In addition, a variety of means...30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular evolution...revolutionize neuroscience Manipulation of specific cell types in neuronal networks Double inverted vectors...combination with mouse lines that express Cre only in the cell type of interest (Atasoy et al. 2008; Cardin et ...

...proteins in ten ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation... small cell populations, co-microinjection of TRIM21 protein and the target antibody works well. In larger...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...larger cell populations, you can deliver these components together using electroporation. In these bulk...bulk cell experiments, Clift et al. observed no stress or damage upon TRIM-Away, and the electroporated...

...look at the subcellular localization of a protein of interest. Conveniently, you found a cell line in which...perform immunocytochemistry on these mutant cells as well as a non-mutant line and see a great antibody...antibody binding in the transfected cells but not in control cells. But (there is always a ‘but’), you...transfect it into your favorite cell line. In parallel, you transfect cells with another plasmid not encoding...the cells transfected with your protein of interest and show just the faintest band from the cells transfected...or may not perform well under the same conditions. Example: You want to sort cells based on the expression...when you start using complex samples such as whole cell lysate or tissue sections. It is important to confirm...

...human T cells and hematopoietic stem and progenitor cells (HSPC)s to ~40% expression in the K562 cell line....201302948 Trapani I, Toriello E, de Simone S, Colella P, Iodice C, Polishchuk EV, Sommella A, Colecchi L, Rossi...gene to airway epithelial cells in mice. This vector transduced ~10% of cells, similar to the transduction...//doi.org/10.1038/mt.2010.205 Trapani I, Colella P, Sommella A, Iodice C, Cesi G, Simone S, Marrocco E... then independently packaged into an AAV. When a cell is co-infected with both part A and part B AAVs,... always.  The combination of serotype and tissue/cell type could have a big impact on the rate of transgene...since both of these variables influence whether cells will be co-transduced with multiple copies of both...

...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...Correctly recombined cells will not contain the negative selection marker, but cells with random recombination...modified version of the targeted sequence to the cell which will recombine the template with the endogenous... used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout...targeting construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required...antibiotic resistance gene. To avoid selecting for cells in which the construct has randomly integrated into...outside one of the homology arms. When we select cells later, we will first perform a positive selection...

...holes in the cell membranes of your sample to allow antibodies better access to intracellular targets. If...allowing antibodies into the cell where they could bind to intracellular epitopes. C) Permeabilizing with...method for visualizing molecules of interest within a cell or tissue. In that introduction, we broke down the...structure of your samples, the relationships between cellular components, and the accessibility of your target...cross-linkers work by (as the name suggests) cross-linking cellular proteins. Cross-linking is a chemical reaction...great for preserving your samples and maintaining cellular and tissue morphology. Formaldehyde is the most...37%). Formalin solutions often contain methanol as well, in order to slow the polymerization of formaldehyde...

...mitochondria reveals dynamic organelle interactions. a) Labeling scheme. b) COS-7 cell ER labeled with Halo-Sec61β...Bewersdorf, J. (2016). Two-colour live-cell nanoscale imaging of intracellular targets. Nature Communications,...better-dyeing-through-chemistry-small-molecule-fluorophores  Los, G. V., Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich...opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure represents...standard confocal microscopy. Image adapted from (Bottanelli et al., 2016) under CC-BY license. Combining... HaloTag for their "ticker tape" method for intracellular recordings, switching between different colored...649-mDia1, magenta) and capping protein (549-CP, yellow) at the tip. 649-mDia1 and 549-CP are both SNAP-tag...

...culture cells in the drug of interest at a lethal dose for non-manipulated cells and then assay cells which...considering either a cellular screen or a selection. A selection is a process in which only cells with the trait...selection, you might put cells into a drug-containing media in which only cells which can tolerate the ... – that is, you are not specifically looking at cells that can survive. Instead, all manipulations are...off. For a screen, you would individually treat cells with many different drugs and then perform an assay...manipulating or mutagenizing a population (typically cells or small organisms) and then assaying for a functional...For example, a drug screen may be performed on a cell line possessing a patient mutation to assess how...