...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...

...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...

...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 ...transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker). Check ...

...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... localization profiles, acting as a biosensor or cell marker, studying protein-protein interactions, visualizing... was non-toxic and could be expressed in living cells, enabling the study of dynamic, physiological processes...monitor gene expression from that promoter in a given cell type. This type of transcription reporting was among...biosensors has been designed to detect a variety of intracellular conditions, including ion (such as Ca2+) concentrations...

...the surface of basophils and mast cells, two types of white blood cells involved in allergic reactions. ... is important because it determines what immune cells and molecules are recruited by the antibody to help...each of these functions: Recruitment of immune cells that express receptors that recognize the Fc portion...which can initiate a cascade that helps attack extracellular pathogens.  Transportation of antibodies to ...special receptors that transport antibodies through cells and into different body compartments, such as into... during a primary immune response because all B cells begin by expressing IgM. IgM has a pentamer structure...coexpressed with IgM on the surface of most immature B cells, its function is unknown. It has a monomer structure...

...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.,... thanks to our guest bloggers Marcelle Tuttle and Alex Chavez! Marcelle Tuttle is a medical student at...Repression and Activation. Cell 159:647–661 . https://doi.org/10.1016/j.cell.2014.09.029 Konermann S, Brigham...Genome-Scale Fitness Screens. Cell Systems 6:555-568.e7 . https://doi.org/10.1016/j.cels.2018.04.011 Polstein ...and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/10.1016/j.cell.2014.09.039 Wong N, Liu W,...Clouse. This post was contributed by guest bloggers Marcelle Tuttle and Alex Chavez, researchers at the Wyss...

... inosine, which is treated like guanosine by the cell, creating an A to G (or T to C) change. Adenine ...

...Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R. Cell Res. 2013 Oct;23(10):1163-71. doi: 10.1038/cr.2013.122...

...dTAG molecule toxicity in new cell lines Treat parental cells (or cells expressing a control -- for example...dTAG system is versatile and can be used in both cell-based assays and mouse models. The dTAG system The...broad utility for degrading targets in different cellular compartments, and in vivo utility. dTAG molecules...of N- and C-terminal fusions and degradation in a cell line of interest prior to starting knock-in experiments...the FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated to date, we observed little...different contexts (for example, IZKF1 and IZKF3 are cell-type specific transcription factors with important...selective FKBP12F36V fusion chimera degradation. In the cell lines we have evaluated to date, we have not observed...

...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... and a desired genetic payload (ex: GFP). Thus, cells can be both sorted using FACS and isolated with ...this system in vitro and in vivo. They identified cells from 19 genera across four phyla that have taken...

...be applicable to studies of cell cycle progression, epigenetics and cellular reactions to external stimuli...Weissman JS, Qi LS, Huang B. Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001. Pubmed. Find...LS, Weissman JS, Vale RD. Cell. 2014 Oct 23;159(3):635-46. doi: 10.1016/j.cell.2014.09.039. Epub 2014 Oct...labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, ...CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique...nuclear organization varies across the life of a cell, or how organization may be altered in disease states...dynamics, and how they change in response to the cell cycle or other stimuli.   To create a colorful Cas9...

...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...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...biosensors for monitoring RTK activation by shift in cellular localization. Image adapted from Farahani et al...

... brain, robust single-cell labeling is necessary to distinguish individual cells. The Brainbow-1.0 construct... May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... both of Harvard’s Department of Molecular and Cellular Biology, harnessed these attributes of Cre-lox... 3.2. Brainbow labeling has also been applied to cell marking and lineage tracing in other systems, such...Brainbow highlights the trend in biology towards single-cell resolution imaging. Sanes and Lichtman endeavored...door for immunostaining analysis. To achieve even cell labeling, they generated farnesylated derivatives...fluorescent proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated ...

...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...

... Viruses are intracellular parasites and natural vehicles for genetic information. Therefore they make...make excellent tools for genetic engineering. There are several different viral vectors to choose from,...Virus (MoMLV, MMLV, MuLV, or MLV) or Murine Stem Cell Virus (MSCV) genomes whereas lentiviral vectors ...things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic diseases...vectors, their infectivity is limited to dividing cells. Find tips on using gamma-retroviral vectors in ...are able to transduce dividing and non-dividing cells. More detailed descriptions of the lentiviral elements...mechanism. Scientists can express siRNAs to direct the cellular RNAi machinery to silence target genes. This empty...

...be expressed. Are you looking at an intracellular or extracellular molecule? By fusing a biosensor to ...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...by Olivier Pertz’s lab. Other biosensors track cellular characteristics like pH, redox state, and voltage...fluorescent signal based on ligand presence or changing cellular conditions. Other types of biosensors To focus...biosensor Before expressing a biosensor in eukaryotic cells, think about the best place for the biosensor to...domain, biosensors can be targeted to a particular cellular compartment such as the nucleus, mitochondria,...

...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...

...manipulations on cells, and has potential use for a wide range of applications in cellular biology. Van ... several cell types is the lack of a rapid and quantitative editing assay. To help accelerate the expansion... of a microtubule-associated protein to regulate cell growth dynamics Article contributed by Klaus Wanisch... by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics with light by modifying EB1. EB1 normally...of the modified EB1 protein are overexpressed in cells, microtubule growth can be switched off with blue...paper demonstrated that the migration of a cancer cell line equipped with photodissociable EB1 could be... opens novel ways in manipulating and studying cellular dynamics. The photoinducible control of protein...

...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...ethanol, work by dehydrating and precipitating cellular components. This method of fixation is good for...