... is technically cell-permeable, its use in live cells can sometimes result in cellular toxicity due to...its propensity to bind cellular components. Some dyes target specific organelles like mitochondria, and... Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental context...Additionally, cell permeability is a critical factor, as it influences the dye's ability to enter cells without...enabled the visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive... dye or ligand to make your selection easier, as well as highlight some recent advancements within this...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...

...this approach is limited to protoplast cells (plant cells whose cell wall has been removed). Another common...expression is dependent upon the cell type in question. In plant cells, gRNA expression is achieved by ...exposed to plant cells. The region of the Ti plasmid between the T-DNA borders (yellow) are recognized ... shown to be excellent alternative genome editing technologies especially in human cells. In contrast ... 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley yellow striate mosaic...are necessary to enable genome editing in plant cells.   This blog post will present a general overview...are necessary to use the CRISPR system in plant cells. Like other model systems, expression of S. pyogenes...

...). These cell-permeable Janelia Fluor (JF) dyes have excellent properties inside living cells, especially... fluorescent proteins. In-cell labeling strategies The majority of in-cell labeling strategies have two...and oligonucleotide labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores..."Imaging live-cell dynamics and structure at the single-molecule level." Molecular cell 58.4 (2015): 644...sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer beholden to chemists and.... Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing fluorophore...question: In this age of GFP and its ilk, why should cell biologists talk to chemists? One reason is something...

...untreated cells—but can we be certain? Do we know whether the plasmid delivered into the cells successfully...test plasmid or transfected into a separate well of your cells. Regardless, the amount of reporter protein...amount of DNA transfected into the cells and the ability of the cells to express the protein.   In analysis...because the purified plasmid, cells, and media used in each of the wells are not independent: they were...Gene X in backbone Y) was transfected into human cells. From this result one could simply conclude that...didn’t work, as the expression level of Gene X in cells treated with Plasmid A is similar to the expression...or transfection process) may have on the target cells. One transfection control is an empty vector control...

...it first infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross...the starter cell, RABV was modified to only infect user-defined starter cell types, as well as to only ...dictate infection of target cells through their interactions with target cell surface receptors (Callaway...Callaway 2008). Based on this, cell type specificity of the starter cell can be achieved by using EnvA-pseudotyped...studied biological processes, like how our stem cells are housed in specific locations and give rise to...Tracers Due to their ability to efficiently enter cells and deliver a variety of cargoes, genetically engineered...replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified Rabies Virus...

...the producer cells and purification protocols often describe a process that begins with cell lysis to release...producer cells into the supernatant during the production phase. However, this has not been well characterized...particles is able to infect more cells (as demonstrated in different cellular environments such as retina ... steps to separate cell culture supernatant containing exo-AVVs from producer cells, and then to enrich...Exosomal vesicles secreted from producer cells also contain cellular macromolecules (e.g. proteins, nucleic...effectively target less accessible cells, such as brain tumor stromal cells in a mouse model of glioblastoma...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector production...

... Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Kanaan, N. M., Sellnow, R. C., Boye... where many different cell types, or even different phenotypes of the same cell type, are intermingled... of the same species. What works well in a mouse may not work as well in a rat or non-human primate. Likewise...postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells in far away...Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan, G., Samulski...experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression is needed... capsids, which determine how a virus enters the cell. Different serotypes utilize a number of different...

...fluorescence images of C2C12 cells stably expressing pTRE-Tight-MitoTimer and rtTA. The cells were pulsed with 2µg...newly synthesized (green) protein and organelles with older (yellow to orange) protein. Image courtesy of... Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1;126(Pt ... targeted to subcellular regions or protein complexes for proteomic mapping in live cells. Lam et al...neighboring cells in imaging applications and the mapping of neuronal projections to their associated cell bodies...been used by several groups to monitor protein and cell ageing, as the spectrum shift occurs during Timer...(pTRE-Tight-MitoTimer) to show its usefulness in cell culture to report changes in mitochondrial turnover...

...activated T cells, precluding analyses of T cell differentiation beginning with naïve T cells. The Framework...screens have been used to interrogate T cells, an important immune cell type, in more complex and physiological...naïve T cells. More broadly, this system can be used to perform in vivo screens in other cell types. A...screens to assess T cell responses and clonal dynamics in lymph nodes (LN) and tumor cells. Figure used with...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine microenvironment that...Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024), developed and deposited...

...molecular level, at the cellular level there is a universal theme - uncontrolled cellular proliferation. We ...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material... directly inhibit the ability of a cell to undergo programmed cell death while simultaneously stimulating...eliminate every cancerous cell. Even so, to pose the greatest threat, cancerous cells still need to successfully...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division. This...proteins that, when expressed, normally encourage cell survival, growth and proliferation, while tumor ...

...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different...separately.  Mutant cells are enriched using either drug selection or fluorescence-based cell sorting and screened...mutant cells can be used in drug screens to identify genes that confer drug-resistance. Mutant cells are ...generate lentivirus (B). Cas9-expressing cells or wild-type cells are treated with lentivirus containing...plus Cas9 to generate the mutant cells, respectively (C). Mutant cells are screened (D) and "hits" are ...lead to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...requires a massive amount of cells as starting material. Therefore, cell types that are of low abundance...

...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...addiction systems” require dividing cells to retain plasmids - if a cell does not inherit plasmid due to ...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...consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the...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 ...

...Cellular communication and function depend on potential energy. Cellular activities like action potentials...Indicators As previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is ...endocrine cells. It follows that the specific targeting of these voltage indicators needs to be cell type ...electrical events! Voltage indicators are voltmeters for cells! In this blog we will review how these indicators...related to the electrical impulse of the cardiac cells. To study this phenomenon you will likely want to...the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by... electrical charges propagated through multiple cells to do things like translate perceived signals to...

...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...RNA aptamers are used to track cellular processes by transforming cells with plasmids encoding aptamer-tagged... been used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ...PMC4244833. Kellenberger, Colleen A., et al. "RNA-based fluorescent biosensors for live cell imaging of...and later introducing the small molecule into the cell. The fluorescence that is generated allows for qualitative...). DFHBI is membrane permeable and non toxic to cells. Because it rapidly photobleaches, DFHBI can only...

... and hexon), as well as other intracellularly-exposed proteins and downstream cellular events, all recruit... AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of the innate immune system... in the producer cells where there is E1 and E3 gene complementation. In target cells that are infected...death of adenoid cells in vitro (Rowe et al., 1953). While overt death of the targeted cells is less likely... & Verma, I. (1998). Cellular immune response to adenoviral vector infected cells does not require de ...infection for efficient cell lysis and release of adenovirus from infected cells. J Virol, 70(4), 2296.... some unprecedented combination of the three (Servellita et al., 2023). Normally when children have acute...

... between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j.cell.2023.05.044. PMID... efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419...lentivirus and MMLV gag, to specific cell types. It can also make CAR-T cell therapy more accessible by simplifying...Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old...called DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design). This modular approach ...viral surface, expanding the range of targetable cell types. DIRECTED is compatible with fusogens from...process and could even enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can ...

...of proteins, organelles, and cells. Similar to FPs, PA-FPs allow for non-invasive labelling, but in contrast...Tetrameric PA-FPS are better for whole cell imaging and studying organelle trafficking. Monomeric PA-FPs are...localization, turnover, and trafficking, as well as organelle trafficking and dynamics (fission, fusion,...offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization,...continuously synthesized and degraded within the cell, so at any given time, an FP-chimeric protein may... reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine...Since emission of green light is more phototoxic to cells, most scientists working on in vivo imaging models...

...the caffeine of the cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:... as proxies for a cellular process or directly measure a cellular process. Cellular phenomena such as ... measure real-time cellular reactions to stimuli and as well as changes in cellular dynamics during so-called...type of cells the viral particles infect. This can direct your biosensor to specific tissues or cell (sub...biosensor is the thing that you care about within your cellular or animal system. This ‘thing’ could be a neurotransmitter...blog we will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost...These ion sensors can be targeted to different cellular locations or tissues depending on the biology ...

... properties and processes with our own cells. Like human cells, yeast DNA is packaged into chromosomes...differentiation of β-cells in the the developing zebrafish pancreas. They first tagged these cells using plasmids...By Alyssa Cecchetelli and Lukas Morgan Model organisms are vital tools used by researchers around the...recapitulate the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than...than studies in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study...to identify a set of mutations that made cancer cells more resistant to chemotherapy. You can find these...to optimally control gene expression in specific cells of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces...

...fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is big...proteins to study mRNA trafficking in living cells." Methods in cell biology 85 (2008): 273-292. PubMed PMID...genomic loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed... fluorescent protein in almost all organisms and cell types, (ii) no need to add a co-factor, (iii) easy...toleration in fusion proteins. Since GFP is such a well-validated, all-round good performing probe, it is...introducing two mutations (F64L, S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant... the bathing solution and require uptake by the cells. Which of these strategies works best for a specific...

...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... starting point, Gaudelli evolved the base editor into 40 new ABE8 variants (Gaudelli et al., 2020). Compared...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual base...editing than ABE7.10 Gaudelli et al., 2020 ABE8e   Efficient editing in primary cells Zhang et al., 2020...., Peraro, L., Young, L., Lee, S.-J., Ciaramella, G., & Gaudelli, N. M. (2023). Improved cytosine base...relatively low frequency, especially in nondividing cells. Scientists everywhere wistfully imagined being ...

...and yeast cells are cost-effective and faster-growing than mammalian cells, but mammalian cells can perform...your cells with your plasmid(s), and then wait 7-14 days (in mammalian cells) to collect the cell culture...production system, cell lines typically used include Human Embryonic Kidney (HEK) cells and Chinese Hamster...An antibody that performs well in immunohistochemistry may not perform well in a western blot. This can...recombinant antibodies with some molecular biology and cell culture experience. Let’s go over the basics of ... place in bacterial, yeast, plant, or mammalian cells. Each of these systems has their advantages and ...post-translational modifications that bacterial and most yeast cells cannot. Your choice of production system may depend...

... trigger the activation of dendritic cells, macrophages, T cells, and the recruitment of neutrophils. ... says. In induced pluripotent stem cells derived endothelial cells from women who are missing an entire...detect molecules outside of the cell and translate those signals into cellular responses. For example, GPCRs...estimated that CTD is the second cause of male intellectual disability after Fragile X. There is no cure...maintaining the nutrient and ionic balance of the cells,” says Dr Sangeetha Iyer, ACD’s Scientific Advisor...that solute channels typically expressed on other cell types can compensate for or bypass the dysfunction...collection to perform a functional screen in mammalian cells to identify any solute channels that can compensate...

...mammalian cells. Here, we will cover how these structures are generated, why it’s important for cells to sense...that doesn’t involve cellular dysregulation) is the mitochondria. These organelles have their own circular...., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat Rev Mol Cell Biol, 23: 286...are the words that describe RNA! Double stranded? Well, that’s just for DNA – right? RNA performs almost...almost all of its biological functions in our cells in the single strand form, but double stranded RNA and...with bidirectional transcription. In an uninfected cell, dsRNA rarely circulates, but upon viral infection...clinical therapy to target pathogenic proteins, as well as in the lab to deplete expression of proteins ...

...copy number in the cell is high, but allows replication when copy number in the cell falls too low (Novcik...system to partition themselves to each daughter cell during cell division. They don’t simply rely on chance...eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When...to an identification issue: the CBPs in the cell can’t tell the difference between plasmids if they have... daughter cell, instead of partitioning each type of plasmid into a separate daughter cells (Figure 2)...from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher...different plasmids to be maintained in one bacterial cell. In this Plasmids 101 post, we’ll cover why this...

...epitopes likely to be presented by T cells to elicit an immune response. T cell epitopes are found in either ...predict T cell epitopes focus on characterizing those epitopes as CD8 or CD4 epitopes, as well as their...that take place within an antigen-presenting cell like a T cell. It is also not yet possible for these algorithms...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of the...CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or macrophages...skin and subcutaneous models (Groell et.al, 2018). Treating these cells with the therapeutic antibody ..., that are commonly used for the prediction of T cell epitopes based on the amino acid sequence of the...

...to monitor cell, organelle, and protein localization. For instance, you might dye your cells and look at...either in whole cells or as a pool of fusion proteins localized to a specific region of the cell. For example...the relationship between the age of a cell, protein, or cellular structure and a particular biological...trafficking to a subcellular location, start of gene expression, development of a cell structure, etc)....biology, FP timers might be able to tell researchers whether old or new cells are better at producing a compound...microscope, fractionate samples to isolate particular organelles and their contents, or perform in situ hybridization...timers, researchers can look at a single image of a cell and understand how protein localization changes ...

...associated with cellular organelles, scientists have been able to study many cellular processes, including...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...Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15(1) (2016...trafficking. Although FPs have enabled discovery of many cellular mechanisms, there are some limitations to working... ultimately misinterpretation of the protein’s cellular role. One way to avoid the pitfalls of overexpressed...synthetic DNA for the desired gRNA target sequence, as well as the 5’ and 3’ homology arm repair template (Fig...

...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively... not well understood, researchers have been using AAV vectors for gene editing in mammalian cells since...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...since 1998. In that year, Russell and Hirata from the University of Washington reported a surprising finding...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...composed of single stranded DNA, which is particularly well suited for strand invasion, a key step in the homologous...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...

...packaging cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not...using helper-free packaging cell lines This method utilizes a packaging cell line that stably expresses... packaging cell line. For example, Phoenix, a second generation γ-retrovirus packaging cell line developed...and rat cells), or an amphotropic envelope, Phoenix-AMPHO (for the infection of mammalian cells.) Using...transduce both dividing and non-dividing cells because once in the host cell cytoplasm, the viral components are...for efficient gene delivery to mammalian cells using 293T cell-based systems. Curr Protoc Immunol. 2001...γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted the development of multiple...