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...expressed in cells that express cre recombinase. Typically, this feature is used to attain cell-specific ...CRISPR applications and can be expressed from your cell line of choice after transduction with this virus...blast virus can be used to generate Cas9-expressing cell lines, which can then be used both for screens (...viral prep to create a single stable Cas9-expressing cell line and then transduce this line with lentiviruses...using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this growth rate effect doesn’t ...activating particular signaling pathways in specific cell types (typically neurons). While all of these viruses...transduction and transgene expression across different cell types (Aschauer et al., 2013). Expression of hM4D...

...plasmid. Envelope proteins determine which types of cells a virus can infect. The VSV-G envelope protein enables... enables this virus to infect many different cell types so it is useful in a wide range of applications... The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR v2...deliver Cas9 and your gRNA of interest into mammalian cells. This vector was the top requested plasmid of 2015... popular in genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using...puromycin cassette that makes it less efficient in some cell lines. pSpCas9(BB)-2A-Puro (PX459) V2.0 was made...capable of inducible expression of shRNA in target cells by the addition of tetracycline/doxycycline to the...

...imaging with single-cell resolution using chemical cocktails and computational analysis." Cell 157.3 (2014):... of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution. To ... optimal expression of XFPs. Any longer and the cells may start to show morphological abnormalities due...XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color variations... wait too long, as too much XFP is toxic to the cells. Inadequate expression levels of XFPs - Reduce the...video below depicting dendrite wiring of mitral cells.   Many thanks to our guest bloggers Richi Sakaguchi...circuitry with an index-optimized clearing agent." Cell reports14.11 (2016): 2718-2732. PubMed PMID: 26972009...

... disease, cell type or drug treatment can browse through screens performed in relevant cell lines or conditions... currently houses over 890 CRISPR screens in 629 cell lines, each manually curated and annotated from ...feasibility and power of CRISPR-based library screens in cell lines from humans and other species. By accurately... screens performed in 26 different breast cancer cell lines. Screenshot updated 2024. Each screen ...screen is captured by BioGRID curators, including the cell line/tissue type, enzyme system, sgRNA library and...example phenotype screens with read-outs other than cell viability. The following video provides an in-depth...

...Localization within cells Fluorescence microscopy Visualize components of individual cells Separation/identification...fluorescently-labeled components Fluorescence-activated cell sorting (for cells); fluorescent HPLC (proteins or other molecules...biosensor Before expressing a biosensor in eukaryotic cells, think about the best place for the biosensor to...average amount of fluorescence across a population of cells (i.e. in a plate well). Use to track a process (... molecules) Isolate or quantify cells, proteins or other molecules that have been labelled with your fluorescent...be expressed. Are you looking at an intracellular or extracellular molecule? By fusing a biosensor to ...by Olivier Pertz’s lab. Other biosensors track cellular characteristics like pH, redox state, and voltage...

...recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo...standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion...recombination efficiency. When expressed in neuronal cells, each line showed broad recombinase-dependent RFP...these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to be functional...functional in cultured Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of ... damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak B, ...recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation. Heidmann...

...eight millimeters – not single cells (yet) but small clusters of cells. Looking ahead What’s next, you...fluorescent proteins to observe what’s happening inside cells for at least a couple of decades. Green is the classic...and tracking of proteins, organelles, and living cells in a spatiotemporal manner, which is not possible... vivo. “One of the reasons we can’t localize one cell at a depth of one centimeter with fluorescence is...on the other hand, are larger than the size of a cell, so they scatter much less. As a result, ultrasound...brighter, more photostable, smaller, and less toxic to cells. They’d like to make near-infrared fluorescent proteins...

...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...transformation of chemically and electrocompetent cells is minimal (picogram range). It can be extracted...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...process of inserting an external plasmid in mammalian cells and it will likely require micrograms of plasmid...

... them into your cells, you can simply look for blue colonies to determine which cells have taken up the... mScarlet as the donor in HeLa cells.   Find ShadowR for mammalian cell expression here   Now that you... that the cellular fitness cost varied greatly between chromoproteins. In some cases, cells containing...cloning experiment worked just by looking at the cells on the plate. For example, if your plasmid contains...Here, the chromoprotein is expressed and gives the cell a magenta (amilCP) or purple (tsPurple) color. Though...added which induces expression of the toxin. Any cells that grow after this point will be those that have...resonance energy transfer (FLIM-FRET) in living cells (Figure 4). They created ShadowR by modifying the...

...Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15(1) (2016...and function of cellular systems. FPs have been used to track protein localization, cell structure, intracellular...to fluorescently tag markers of cellular structures in mammalian cells. These plasmids use fluorescent...loci in other model systems, including mammalian cells. CRISPaint (CRISPR-assisted insertion tagging)...C-terminal tag fusions of endogenous genes in human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA...also be introduced directly into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers...selection. Researchers at the Allen Institute for Cell Science recently developed a collection of plasmids...

...ancestry and germline sequence variation in cancer cell lines took them down unexpected paths. Though this...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of...to see if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American...or South Asian) used in the screens based on the cells’ germline genetic variations. They then wanted to...with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either...groups for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of...CRISPR guides, short pieces of RNA that bind to a cell’s DNA, telling the Cas machinery where to cut.  ...

... into AAV particles for delivery into mammalian cells, especially when fused to effector proteins for ..., base editing, and genome editing in mammalian cells. The “V4” version (named CasMINI) had the highest...an active nuclease.  Read the paper in Molecular Cell Find the CasMINI plasmids Efficient CRISPR editing...effective in genome editing in both bacteria and human cells using various delivery methods, including plasmid...mRNA or Cas9 mRNA and sgRNA, in human and mouse cells. They further develop a version of SEND for gene...modular system for delivering gene editing tools into cells. Image adapted from Segel et al. 2021.   Prime...correct mutations in a variety of target genes and cell types. Their Nature Biotechnology paper showcases...

...of Multiple Kinase Activities in Live Single Cells." Cell 157, no. 7 (2014): 1724-734. PubMed PMID: 24949979...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ... dimeric Dronpa is less prone to aggregation in cells compared to the tetrameric Dronpa it was derived...inhibitors, psMEK1 and ERK KTR-mRuby2 expressing cells were incubated with an inhibitor of choice, exposed...inactivation, and then decreases. Treatment of the cells with a PP1/PP2A inhibitor leads to maintenance of...Raf-MEK-ERK signaling in the hypodermis and gut cells of C. elegans causes the same phenotype. Activation...signaling pathway with psRaf1 or psMEK1 in these same cells by 24-48 hours exposure to blue light also induced...

...intensity evolves over time in living cells at a power used for live-cell imaging. To conclude, the most photostable...molecule will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins...experiment. The best fluorescent proteins for live cell imaging can be excited many times, thereby producing...protein is a key requirement for quantitative live-cell imaging, it is important to be able to quantify ...it is advisable to perform time-lapse imaging of cells producing the fluorescent protein of interest under...natural situation. Measuring photostability in living cells provides a much more realistic view of photostability...spectral class on the microscopy system and in the cell type (or tissue) that is intended to be used with...

... protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of...disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant...which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB...broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon...Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly...can one work with a plasmid/gene that kills the cells expressing it? The answer lies in special strains...PMID: 8254658. 5. Bernard, P. and Couturier, M. “Cell killing by the F plasmid CcdB protein involves poisoning...

...Purification Process with pCri Cell-free protein expression kit Cell-free expression systems have been...testing and comparison of different cell-free expression systems; the Cell-free expression test kit represents...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of...: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression Kit FREQ Seq PUF Assembly...facilitate gene expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression...of proteins in both prokaryotic and eukaryotic cell extracts (Gagoski et al 2015). He has also constructed...to perform statistically relevant benchmarking of cell-free expression systems, and tested for product ...

...percentage of total live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive ...would serve you well. Percentage of cells in a parent gate (all live cells for example) that are positive ...example, you have introduced a GFP protein into your cells (visualized via the FITC channel) and you are trying...graphs could simply be the percentage of the total cells present in the gate.   Figure 1: Example...looking at proteins that are expressed regularly in cells and might be upregulated or downregulated based ...populations. MFI is the average brightness from all cells that are positive for the marker of interest. MFI...

... to express one gene in one cell type and another gene in a different cell type (6–8). Although it’s often...systematically study gene function with temporal control and cell-type specificity! The Gal4/UAS system At a more...the hedgehog gene can be overexpressed in several cell types by using different Gal4 drivers. It is much...much easier to mate flies to get the right cell type expression as opposed to making a new fly with hedgehog... Targeted gene expression as a means of altering cell fates and generating dominant phenotypes., Development...Neuro-Trauma during Asymptomatic Viral Infections, Cell Host Microbe 22, 48-60 (2017). PubMed PMID: 28704652...expression, lineage tracing, and mosaic analysis., Cell 141, 536–48 (2010). PubMed PMID: 20434990. PubMed...

...splitting my cells first. Time management through planning I probably wouldn’t have fallen into my cell splitting...for Scientists. Back when I used to do mammalian cell culture, I would often respond to friends asking...there in a few minutes, I just need to split some cells.” Inevitably, this task would take longer than I...since my days as an undergrad working with cancer cell lines. As I started planning out my own experiments.... In my anecdote above, I prioritized splitting cells over meeting up with friends, but didn’t realize...mean in terms of the utility of your tasks. In my cell splitting anecdote, I probably could have grabbed...with friends and come back to the lab to split my cells later. Eating with friends has a somewhat immediate...

...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...energy source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins...