...neuroscience where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...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...

...scFvs and T cell receptors and are expressed on T cells to make CAR T cells. The extracellular scFv portion...Bispecific T-cell engagers (BiTE®s), bind CD3 on T cells and a tumor-specific antigen, it brings T cells to a ...recognizes an antigen of interest while the intracellular T cell receptor portion of the CAR facilitates ...scFv part of the receptor can activate T cells to kill cancer cells. scFv’s small size also allows for their... display libraries screen antibody genes from B cells of immunized animals, but it's possible to avoid...also allows for screening scFv generated from the B cell antibody genes from diseased or vaccinated humans...produce scFvs in mammalian, yeast, plant, and insect cells, they are most often expressed from a plasmid in...

...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...Stop and smell the flowers - or rather, the lactate! The latest biosensor from Gary Yellen’s lab is LiLac...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...

...Gordon Fishell New viral prep pAAV_BiSSTe10_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiCHATe27_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiVIPe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiLAMP5e3_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiPVe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiPVe3_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...Gordon Fishell New viral prep pAAV_BiSSTe4_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral...

...co-expression among cellular markers You’ll also need to know if markers are co-expressed in your cells when designing...immunologists, allowing for the rapid analysis of cell populations within a heterogenous tissue type, such...methods, the identification and isolation of rare cell types. In recent years, full spectrum flow cytometry...detector to detect one fluorophore and therefore one cellular marker. However, emission spectra of certain fluorophores...Spectral flow allows for autofluorescence extraction Cells produce a low level of fluorescence called autofluorescence...your panel. If you have two highly overlapping cellular markers, it is important to pair them with two...similarity index of 0.87; therefore, two overlapping cellular markers should not be paired with these dyes, ...

...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...issues with tissue autofluorescence, cell heterogeneity, and/or cell position changes (To et al., 2016, ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP...cleavage by executioner caspases. Failure for cells to die or cells dying at the wrong time and/or place can...Apoptosis or “programmed cell death" plays a pivotal role in an array of biological processes including...including development, the immune system, and cell turnover. Apoptosis is a highly controlled process that ...sfCherry however did not initially fluoresce in HEK293 cells. To rectify this the Shu lab first improved the ...

...Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Molecular Cell 9:789–798. https... Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent protein, YFP) is reunited by the interaction...directly visualize PPIs that regulate numerous cellular functions, including ubiquitinoylation, kinase...BiFC studies used the method to interrogate the subcellular localization of interactions between basic leucine...fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. The straightforward...involves three steps: fusion protein construction, cellular expression, and quantification (Kerppola, 2006...Fusion Proteins - Making the Right Connection. Cellular expression Whether BiFC constructs are expressed...

...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...after transfection, and are quickly cleared from the cell via protein degradation pathways. This makes Cas9...

...mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced cells. Grant...in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org/10.1016/j.cell.2016.08.056 Find plasmids...targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from...the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general...pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression...for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD ...BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP...

...dodge cellular variation observed in mammalian cells is to tag an endogenous gene. By imaging cells (or ...mammalian cells within the dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct...under your experimental conditions. This is time well spent and, if you do it right, can be (part of) ...comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein...strongly depends on both the biological system as well as the imaging method, we recommend basing your ...intended application. The three key properties for live cell imaging that will be treated in this series of posts...with a high intrinsic brightness that does not fold well or cannot be detected in a microscopy setup has ...

...bottom of Tokyo Bay. When a weird, sulfurous smelling, black pellet was sent to the AbuGoot lab for testing...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...turning potential into impact. In molecular and cellular biology, this happens through the development ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two...or DNA via a CRISPR mechanism. The weird black pellet's DNA yielded a new CRISPR effector protein they...

... Alcaligenes eutrophus, Bartonella bacilliformis, Bordetella spp., Brucella spp., Caulobacter crescentus...Hyphomicrobium spp., Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium...rubrifaciens, E. stewartii, Escherichia coli, Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus...trifolii, Salmonella enteritidis, S. typhimurium, S. ordonez, Serratia marcescens, Shigella spp., Vibrio...ability to establish and maintain itself within host cells.  The plasmid has multiple ori that are functionally..., Azospirillum braziliense, Azotobacter spp., Bordetella spp., Caulobacter spp., Enterobacteriaceae, Gluconacetobacter...xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus...

... a big issue for cells, but it may confound research results or even kill your cells if your gene of interest...necessary to replicate plasmids and make sure the cells maintains it. Here, we'll cover the promoter, which... Because transcription machinery differs between cell types or organisms, promoters must be similarly ...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique...complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on... at will with a chemical, heat, or light. In the cell, promoters themselves are controlled by still other...

...genes known to be essential for cell growth. Together, this excellent work highlights the importance of...fluorescence-activated cell sorting (FACS). This is not possible in plants outside of limited, single cell protoplast...exquisitely and robustly self-organize at the cellular and multicellular scale.     References Bezanilla M, Perroud...Generation. Plant Cell 24:1478–1493 . https://doi.org/10.1105/tpc.112.098509 Smits AH, Ziebell F, Joberty G...can be found at Addgene that could serve as an excellent start (#22988, #137884). Both vectors allow the...2018) and the model bryophyte Physcomitrium (Physcomitrella) patens (Bezanilla et al., 2005; Nakaoka et...visually identified subpopulation. For traditional cell culture, enriching your active RNAi subpopulation...

... into AAV particles for delivery into mammalian cells, especially when fused to effector proteins for ...size of the Cas9 or Cas12a based systems, making it well suited for AAV packaging. Most importantly, this..., 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...activity and decrease the gRNA size by intensively remodelling the gRNA scaffold through five rounds of gRNA...effective in genome editing in both bacteria and human cells using various delivery methods, including plasmid...plasmids   Selective endogenous encapsidation for cellular delivery of RNAs By Mike Lacy The retroviral-like...

...Purification Process with pCri Cell-free protein expression kit Cell-free expression systems have been...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of...testing and comparison of different cell-free expression systems; the Cell-free expression test kit represents...: 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...combinations of protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein purification...of proteins in both prokaryotic and eukaryotic cell extracts (Gagoski et al 2015). He has also constructed...

... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...we will explore some of the most commonly used as well as newly developed calcium sensors. Selecting a...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ... violet light and (2) exposure to increased intracellular calcium. It has been applied to mark active ...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version named...neuron-specific, astrocyte-specific (GCaMP6 only) or mammalian cell expression, and compatible with constitutive or ...

...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...Read on for an overview of the titering options as well as the benefits and drawbacks of different methods...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...

...reagents and keep the cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and...however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend...particles that can transduce cells, which can vary based on target cells and can be altered by freeze/...several facilities nationwide where you can obtain excellent quality, high titer rAAV (including Addgene!),...come into contact with the rAAV and apply common cell culture practices diligently throughout the production...capsid genes.  In this helper-free system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with...and then collect the supernatant and/or producer cells for further processing. What component(s) you need...

...at bioRxiv. AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral...of the packaging cells (Vandenberghe, 2010). Rather than being released into the cell culture supernatant... particles associate with the cell membrane and are taken into the cell via endocytosis. In our study,...AAV2 viral particles by our packaging cells by treating the cells with the endocytosis inhibitor sucrose...target cells is blocked following sucrose treatment. Moreover, several reports have shown that cells treated... yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype specific...difference between untreated and sucrose treated cells. Although we were unable to deduce the mechanism...

...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat the issues... detects the widely used HA epitope in diverse cellular environments. They called this probe the HA frankenbody...previously proven to fold and function in living cells. To make these scaffolds specific to different targets...probe structure and stability from the scaffold as well as specificity towards a HA epitope from the CDRs...the tagged protein upon probe binding in living cells.  In their proof of concept experiment the Stasevich...mCherry, 4xHA tagged histone protein (H2B) in U2OS cells. They then showed that the HA frankenbody probe ...

...agent than BME and has a less… distinctive smell. BME smells like rotting eggs but has a longer shelf life...expensive Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased...Both polyclonal and monoclonal antibodies can work well for a western blot. Polyclonal antibodies, which...optimization and likely higher background noise as well, and multiplexing is challenging and limited. Direct... mechanical method before your enzyme lysis. For cell cultures, enzyme lysis is typically sufficient.  ...When choosing your lysis buffer, consider the subcellular location of your protein. Proteins located in...proteins easily available in the cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, ...

...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with strong...High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences ...imaging or flow cytometry applications in mammalian cells. The modules used in this method include fluorescent...components, one can generate constructs to visualize cell cycle dynamics, titrate inducible transgene expression...detection of each fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave...

...purify DNA, cellulose also efficiently binds, or at least entraps nucleic acids. The Botella lab took advantage...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave...four methods for DNA purification without a kit as well as one way to reuse silica columns from DNA purification...glass. After binding DNA, the glass particles are pelleted by centrifugation and washed before DNA is eluted... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant... plant, microbe, blood, or mammalian cells or tissue Product: total nucleic acids While glass or silica...

... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...minimal side-effects on healthy cells and maximal effect on unhealthy cells. Until recently, the human genome...ENCODE project, find enhancers in embryonic stem cells, and conclude with a targeted CRISPR screen that...essential transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant...We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus...The result is a heterogenous population of stem cells, usually housed in a single flask. Through a bit...

...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...CRISPR in adult stem cells has huge potential, because the genomes of adult stem cells in organoid cultures...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank...connected to the early onset of Alzheimer’s disease, as well as those that play into late onset AD (5).     ...homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (8).   The difference...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis....is a big advantage over induced pluripotent stem cells, which need to go through a phase of crisis where...

...Susceptible Subcellular Imaging Improving Well-established High-throughput assays Improving Well-established...luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver ...miRNAs for effectiveness. Analyze Cell Signaling Pathways Known cellular signaling pathways can be investigated...used bioluminescent reporter. This beetle emits a yellow-green light with a peak emission at 560nm. Shortly...luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since become a gold standard... serves as an indicator of transcription within cells, where detection of the reporter protein or its ... into the desired mammalian, plant or bacterial cells. Expression of luciferase as the reporter gene is...

...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...an inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...follow the same initial mechanism as in mammalian cells — by excising the freshly edited U bases — but E...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. Beyond...

...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...collateral RNA cleavage response is well described in bacterial cells, LwaCas13a does not display nonspecific...eukaryotic cells. They arrived at this finding through analyses of global RNA expression, cell stress, and...they identified 53 potential candidate genes that fell into 3 categories based on the architecture of the...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...pool of RNAs) or studying RNA processing in live cells. Indeed, groups have utilized dCas13 fusion proteins...genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID-19 (Joung, Ladha...

... immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new approaches to reprogramming... fields of Cancer, Inflammation and Immunity, as well as various combinations thereof (so called “Immuno-Oncology...cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer, Inflammation and Immunity and...immunosurveillance. The presented study analysed a model of hepatocellular carcinoma, a cancer which can develop from ...example the upregulation of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment...follow the interactions between tumor and immune cells in real time. Addgene depositor Professor Max Krummel...effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system - COX inhibitors like aspirin...