...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...

...lets AAVs “unlock” and infect specific cells. This protein shell packages the vector’s genetic material...particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor and sometimes ...transduction efficiency, and is an excellent choice for targeting cardiac cells (Bish et al., 2008). It can ... Therapy: An Overview. Cells, 12(5), 785. https://doi.org/10.3390/cells12050785 Kaludov, N., Brown, K....delivery across species. Cell, 184(19), 4919-4938.e22. https://doi.org/10.1016/j.cell.2021.08.028 Tervo, D...tissue tropism, allowing them to target particular cell types. Figure 1: Diagram of AAV components...multiple coreceptors, which may be present on different cell types and sometimes vary between target species ...

...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, ...

...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...

...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...

...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...

...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...

...the surface of basophils and mast cells, two types of white blood cells involved in allergic reactions. ...determines several key characteristics of an antibody as well as the role it plays in an immune response. But ... 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...isotype nomenclature. Antibody heavy chain proteins as well as the genes that encode those proteins are designated...

...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...

...within an E. coli cell, but vectors can be widely utilized across many different cell types and each one... the yeast cell. These elements control not only the number of plasmids found in each cell, but also whether...are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals... ease of replica plating and mutant isolation, a well-defined genetic system, and a highly versatile DNA...diploid state which is useful for genetic analysis, as well as an efficient mechanism of homologous recombination...components to allow shuttling between E. coli and yeast cells. To be useful in the lab, the vectors must contain...replication (ORI) and a means of selection in yeast cells, in addition to the bacterial ORI and antibiotic...

...development. Single cell sequencing-based lineage tracing Recently, gene expression in all cells of the developing...gene activity and cell divisions that coordinate development of the body plan (Farrell et al., 2018). This...proportions of otherwise normal cell types are produced, rather than novel, abnormal cell types. Sequencing-based...): 193. PubMed PMID: 30944477. Farrell, Jeffrey A., et al. "Single-cell reconstruction of developmental...applications. For example, specific tissues such as blood cells or neurons can be fluorescently labeled, allowing...Strategies for generating transgenic zebrafish are well established (Felker and Mosimann, 2016). One common...divisions, and movements of each of the thousands of cells in the developing zebrafish embryo have been tracked...

...dTAG molecule toxicity in new cell lines Treat parental cells (or cells expressing a control -- for example...contributed by guest blogger Behnam Nabet, a postdoctoral fellow at Dana-Farber Cancer Institute. Targeted protein...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...

...for neurons. Non-neuronal cells do this too. It's just that for non-neural cells, ion-based communication...between many cells (through gap junction proteins which connect the cytoplasm of two cells) can also affect...more depolarized (positive) the cell is, the more embryonic, stem cell-like, or cancerous it is (Levin...happens at a slightly slower scale.  All living cells with a membrane possess an electrical state. This...charge between the inside and the outside of the cell. This imbalance is maintained by ion channels and...when I attended the Developmental Bioelectricity satellite symposium at the 2019 Society for Developmental...proteins cx43 GJB1 GJB2 Bioelectric states can alter cell types and patterning There is mounting evidence ...

...to us? Well, an AAV user must determine which serotype is most efficient at transducing the cell type under...optogenetics as tools to investigate the roles of certain cell types in locomotion. We have relied heavily upon...deliver chemogenetic/optogenetic constructs into our cells of interest. Although they have a small packaging...of serotype (which determines which tissues and cells an AAV can infect) can influence an AAV’s value ... tissue? Location (2): Where in the tissue/which cell type? Location (3): Traveling from the original ...AAV5, AAV6, AAV9 Pancreas AAV8 Photoreceptor Cells AAV2, AAV5, AAV8 RPE (Retinal Pigment Epithelium...-vectors/aav/aav-guide. Where in the tissue/what cell type? In the CNS, for instance, there are marked...

... 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 ...up of separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe ...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...

...yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein...post: Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic...antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR...CRISPR technology over the past decade, primary cells remain difficult to genetically edit due to toxicity...toxicity from electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed...Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an ...including in clinically-relevant lines such as CAR-T cells. Plus, this method is fast! These editing frequencies...

...Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides to a substrate Flow...carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier...chemically. Enzymatic biotinylation occurs naturally in cells and is the process by which biotin is covalently...sequestering biotin from being absorbed. In 1940 Esmond E. Snell was able to purify the protein responsible for binding...the chicken, “Avian”, egg it was purified from. Snell later confirmed that avidin was indeed the culprit... for use in immunohistochemistry and antibody labelling. In these experiments, biotin is added to a primary... Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for biotinylating...

...Shcherbakova et al., 2016), as well as in four-color imaging (cyan, yellow, red, and near-infrared) (Qian...reporters and biosensors NIR reporters can tell us about cellular events via  fluorescence changes occurring...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...

... Mixing the subclones within a single well reduced well-to-well variability, while the readout was able.... Nat Cell Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal...with one visual barcode per cell and up to 200-plex with two barcodes per cell. The researchers validated...long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for ... powerful approach for proteomic mapping in both cell culture and whole organisms (see our 2019 overview...reduced risks of unwanted mutations in mammalian cells. Long sequences traditionally have higher risks ...multiplexing by: Rachel Leeson Multiplexed images in live cells remains a challenge due to multiple factors, including...

... N1 Yellow Mammalian Express a gene of interest fused to the N-terminus of Amber Amber C1 Yellow Mammalian... pCEP4YPet-MAMM Yellow Mammalian Expresses mammalian optimized YPet pYPet-His Yellow Bacterial Expresses...(FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. ... The student was of course talking about the now well-known FRET, aka Fluorescence (Förster) Resonance...measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions...suitable for studying interactions within living cells. By coupling fluorophores to proteins, pioneers ...directly detect protein/protein interactions in living cells. Since then FRET has also been used to measure conformational...

...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...application of AAV is delivery of genetic cargo to a host cell. In order to make room for a gene of interest, AAVs...Serotype refers to the type of capsid (outer protein shell) used to package an rAAV vector; these capsids allow...allow the virus to infect (or not infect) different cell types with some specificity. There are a lot of ...

...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...dead Cas9 (dCas9) has many applications, the most well-known being transcriptional activation/repression...

...illumination of neighboring cells evokes action potentials (APs) in patched cells expressing wild type CoChR...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here...28646204. soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego ...wild type CoChR, soCoChR localizes primarily to the cell body with very little signal detected along the ...