... to environmental conditions, cells must be able to detect extracellular stimuli. One way they do so is...complex converts a signal received by a GPCR on the cell’s surface into action that can result in the initiation...protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing the GPCR... and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the luminescent enzyme substrate...updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric G protein dissociation...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...number of signaling cascades, inducing different cellular responses. One of the biggest advantages of such...

...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...will be degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain...10% of cells while plasmids containing the plasmid addiction system were retained in 75% of cells. They...generations (Figure 1). These so called “plasmid addiction systems” require dividing cells to retain plasmids...plasmids - if a cell does not inherit plasmid due to improper plasmid partitioning, it will still inherit...designated this fragment as the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists...cleaved complex. This results in DNA breakage and cell death. Conversely, CcdA binds to and blocks the ...

... into a mouse embryonic stem cell (ESC) line, enrich and select for cells that had successfully incorporated...gets exciting – the cell sends out an “SOS” signal and cellular repair mechanisms rush in to repair the...Cas-Mediated Genome Engineering. Cell 154:1370–1379 . https://doi.org/10.1016/j.cell.2013.08.022 Yoon Y, Wang ...Cas-Mediated Genome Engineering. Cell 153:910–918 . https://doi.org/10.1016/j.cell.2013.04.025 Zhang X, Liang...develop into chimeric mice (with a fraction of the cells in the adult mice derived from the engineered ESCs...Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...

...proteins in cells. Homeostatic regulation of protein abundance is essential for normal cell function, and...one particular protein in neurons or in any other cell type. However, we cannot distinguish new from old...facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed from...techniques to follow translation in living single cells. However, before CRISPR-Cas9 these techniques were... turnover of specific endogenous proteins in any cell type. Exciting! Many thanks to our guest blogger... how small molecule fluorophores can be used for cell labeling Check out other protein tagging strategies... contact her at [email protected]. References 1. Dieterich, Daniela C., et al. "In situ visualization...

...that it appears “newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand..., which may lead to cell death rather than NHEJ repair, thus removing these cells from the edited population...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual base...relatively low frequency, especially in nondividing cells. Scientists everywhere wistfully imagined being ...editing efficiency, Komor and team wanted to force the cell to use the deaminated DNA strand as a template. ...frequency to above 30% for a variety of targets in human cell lines, with an average indel frequency of only 1.1%...modifications. CRISPR base editing persists through multiple cell divisions, indicating that this method produces ...

... how it works. The findings in a recent issue of Cell demonstrate how PGC-1a1, a transcriptional coactivator... in a commentary about the new work published in Cell Metabolism. But, they note, there is plenty more...’ve come to the right place. Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle...depression, and now researchers have reported a mechanism based on studies of transgenic mice that helps...JR, Erhardt S, Lindskog M, Ruas JL. doi: 10.1016/j.cell.2014.07.051. PMID: 25259918 Muscle over mind. Moon...

...tag and small molecule. It does work in mammalian cells, mice, and even yeast, though (we have a kit!). ...and complete degradation of proteins in mammalian cells. Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):E3350...Tags Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small ... tag size, which can also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V...CRISPR, siRNA, etc. Firstly, they are reversible mechanisms of control. Protein degradation can be induced...uses a small molecule, a tag, and the endogenous cellular degradation machinery to function. The dTAG is...

...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...’ll likely want to use a mechanical method before your enzyme lysis. For cell cultures, enzyme lysis is...proteins easily available in the cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, ...while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the proprietary...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...

...vectors (right) are transduced to target cells. Within target cells, Cas9 machinery is transiently expressed...in order to facilitate its entry into the target cells (Beatty et al., 2012). These features make AdV an... complexes into transformed and non-transformed cells yielded rates of targeted mutagenesis similar to...nuclease-induced gene disruption frequencies in the various cell types ranged from 18% to 65% (Maggio et al., 2014... leads to a vast majority of AdV-modified human cells being subjected to scarless homology-directed genome...therapeutically relevant, non-transformed mammalian cells. These viral vector systems also work equally well... dividing and quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral ...

...successfully infected/expressing cells by flow cytometry. The isolated cells were injected into mice and imaged...to changes in intracellular calcium and is useful for direct imaging of events in cells and brain explants...packaged into a retrovirus for infection of host cells. The fluorescent moiety of the BRET reporter was...imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs) can be problematic...available through Addgene (Plasmid #83926). Live-Cell Voltage Sensor  Ingaki and colleagues (8) were interested... in supporting Promega cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...

...multiple plasmids in one cell, it might be that you can't stably maintain them in the cell. This blog post features...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...features two mechanisms that cause plasmids to be incompatible. 4. RNA Extraction Without A Kit by Leah...

...neuroscience where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted...disease, neuroscientists need to be able to probe cells and circuits without disrupting normal brain function...it provides a noninvasive method for manipulating cell activity, especially neuronal firing. What is chemogenetics...drugs. Once activated, chemogenetic receptors hijack cell signaling pathways and can lead to such responses...requires successfully introducing receptors to target cells. Targeted and specific delivery becomes especially...ability of agonists to successfully reach target cells while resulting in minimal off target effects. For...can elicit different responses depending on what cell type it is expressed in. The most common target ...

...no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7-11...such as mouse embryonic stem cells and glioblastoma cells. PASTE Development and discovery The first ...observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s intrinsic machinery... shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same comparisons, the AbuGoot...11. This included tests done in cell lines known to be more sensitive to toxicity from RNA editing, such...integrases with efficient integration activity in human cells. Moreover, they optimized the specific attachment... of plasmids, works in non-dividing and primary cells, and can be easily programmed for new genes. In ...

...mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas...present in most of the cell culture facilities, tissue culture labs and every cell culturist has to deal...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of... should be made available for cell culturing and maintenance of the cell lines. Providing such an environment...Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma contamination...cryopreserved vials of the cell lines even though the viability of cryopreserved cells decreases with every ...cause mycoplasma infection from one cell line to another. Multiple cell lines should not be stored in a single...

...post-doc a Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial...would continue on with my academic dream of studying cell division. However, after 6 years in the business...information on ELISA troubleshooting, ELISA kits and cell death assays. You can find Seán on twitter @Macfhearraigh... After all I was only going to be interviewed for 1 hour so having a broad enough insight was going to...

...into the cytoplasm, migrates out of the cell, and binds cells that can begin an immune response. This ...Keytruda) causes T-cells to more easily recognize and cause the destruction of cancer cells. Pembrolizumab...high mobility group box 1 protein. Normally localized to the nucleus, if the cell dies from injury, this...cause inflammation, which can cause mesothelial cells to mutate and ultimately form cancerous tumors.  ...resection), systemic chemotherapy to kill cancerous cells that may have spread beyond the point of origin,...successful in killing many (if not all) of the cancer cells that may be left behind after surgery, while being...of immunotherapy is the use of drugs (or other mechanisms) that work through body’s immune system to fight...

...predict that we’d be able to edit RNA in mammalian cells with CRISPR? I certainly didn’t, but now we can ...Improve HDR Efficiency for CRISPR Editing in Human Cells Day 3: Cpf1: Cpf1: A New Tool for CRISPR Genome ...social media? No problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR... Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous End Joining and PITCHing...

...slightly more efficient and less toxic to mammalian cells (Allen, et al., Frontiers in Genome Editing).  Some...enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33, 985–989 (2015). https://doi....Enhance CRISPR Genome Editing Systems in Mammalian Cells. Frontiers in Genome Editing, 28 (2021). https:/...deactivation with photocleavable gRNAs. Molecular Cell 81, 7, 1553-1565 (2021). https://doi.org/10.1016...incorporated at the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability...evolved ways to prevent degradation of important cellular RNA molecules with post translational modifications...expression systems are constantly “on”. There is no mechanism to turn cutting off or on at a specific time. ...

...are often used to study vertebrate embryology and cell development. One major resource for frog research...Organism to Study and Model Human Genetic Disease. Cells Tissues Organs, 205(5–6), 303–313. https://doi.org...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...& Jacob, H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm...

...the mesocorticolimbic system in addiction. Front Cell Neurosci. 2014;8:466. PubMed PMID: 25653591. PubMed...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky... confirms the previously suggested inhibitory mechanism of locomotor sensitization and might open new ...