...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...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...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a viral ...

...for labeling specific structures within cells. HeLa cells were transfected with plasmids encoding...protein evolution Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system...mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image from Mateo...labeling structures and compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent...or specific localization of biomolecules within cells. However, sometimes these fluorescent proteins might...exactly a structure or protein is located within a cell. The Goedhart lab, Gadella lab, and collaborators...for labeling a list of specific structures within cells. For example, to improve nuclear localization of...

...targeted One Many Sample type(s) Cell lines Cell lines, primary cells Table 1: Key differences between... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...need to create stable cell lines and allows CAPTURE 2.0 to be used with primary cells. Find plasmids for...that occur during development and cellular differentiation. Using a cell line model, the Xu Lab tracked ...by Biotinylated dCas9. Cell 170:1028–1043.e19 . https://doi.org/10.1016/j.cell.2017.08.003 Liu, X., Chen...CAPTURE method requires the creation of a stable cell line that co-expresses three components: dCas9 ...drawback of the original CAPTURE method is that a new cell line has to be created for each genomic loci of ...

...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 ...

...in live cells is that one can now follow movement of two or more targeted loci during the cell cycle or...mammalian cells, yeast, and Drosophila. Synchronization of secretory protein traffic in mammalian cells The...populations within the same cell. Mitochondrial fusion is typically assayed in living cells by using mitochondrially-targeted... alternative splicing in mammalian cells. Younis, et al. Mol Cell Biol. 2010 Apr;30(7):1718-28. New...yeast cell viability, had less of an effect on GFP diffusion, properly localized in mammalian cells without...fluorescent fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61...Friedman, et al. Mol Biol Cell. 2013. PubMed PMID: 23389631 Rowland, et al. Cell. 2014. PubMed PMID: 25416943...

...hijacking the machinery of the cells they infect. Once they enter a cell, they treat their viral genome...genome as a “payload” that is delivered to the cell. The cell then uses its own machinery to replicate the...replication process whereby one cell copies itself and “splits” into two cells. Viral vectors Viruses are ...virus. From one, come many After a virion infects a cell, the single particle creates many copies of itself...millions of new copies, which are released from the cell like baby spiders from an egg sac. (Okay, some enveloped...lysing, but they do generally still kill the infected cell.) This is in contrast to bacteria and eukaryotes...means the vector can deliver a genetic payload to a cell but cannot create new particles. As a bonus, this...

...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...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...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 ...

...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...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...determines plasmid copy number within the bacterial cell. Similary, the specific ORI elements included within...plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors can also be controlled...

...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...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...even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40...

...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...investing time and resources into making a stable cell line. When expressing a viral construct transiently...

...specific cell type rely on transgenic models that express recombinases like Cre or Flp in their cells of interest...entry into the cell (Dudek, AM. 2018).  If the serotype you are using transduces cells that you are not... an ATG start codon to prevent transcription in cells where the recombinase was not present (Kuhlman, ...recombination that can lead to leaky expression in cells that do not express Cre or Flp. Here at Addgene ...recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally, we have experienced.../Flex vectors and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about...volume needed to achieve adequate expression in your cells of interest.  The AAV serotype used can exhibit ...

...effectors to visualize subcellular positioning of target DNA sequences in living cells (TALE-mediated Genome...serial ligation. Validated in zebrafish somatic cells. Keith Joung Zhang Lab TALE Toolbox PCR/Golden Gate...ligation-independent cloning (LIC). Validated in human cells. Veit Hornung Musunuru/Cowan Lab TALEN Kit Kit comprised..., both in microinjected embryos and transfected cells. TALEN expression is driven by the strong CAG promoter...destination vectors and a reporter vector for mammalian cell-based validation assay that are designed for use... the evaluation of TALEN activities in cultured cells. Golden Gate TALEN 2.0 47388 pcDNA3.1-GoldenGate...in mammalian systems including hard-to-transfect cell lines. Features include (i) Esp3I (BsmBI) restriction...

...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... the bathing solution and require uptake by the cells. Which of these strategies works best for a specific...showing that its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles... This approach enables the study of proteins in cells, but not other biomolecules of interest e.g. DNA...several components that are naturally present in cells or media. These sources of ‘autofluorescence’ include...

...tensions before they would enable cells to spread, resulting in cells that elongated and morphed asymmetrically...on the BJ-5ta fibroblast cells, integrin αVβ1 was critical in initiating cell spreading and two other ...linkage of intra- and extracellular environments. Found on nearly every cell in the human body, integrins are... that protrude from the cell membrane, physically connecting the extracellular environment and the cytoskeleton...ligand binding, mediating cell adhesion and migration, initiating intracellular signal cascades, and allowing... the extracellular matrix. During immune responses, this same conformation encourages immune cell adhesion...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...

...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...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...system was employed to evaluate degradation of ENL. Cell lines were engineered to express ENL-FKBP12F36V ...

...gets interrupted as shown in cell A, and thus will not complement the cell's β-galactosidase mutation. An...therefore the cell will have a functional β-galactosidase enzyme through α-complementation (cell B). As shown... plasmid-containing cells without using antibiotic resistance. These rely on cell lines susceptible to...without which the cells cannot reproduce to form colonies (used to complement auxotrophic cell lines), or genes...called the α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a...screening process. It does not select only those cells that have taken up a plasmid and thus should be ...white due to successful cloning and not because the cell failed to take up the α-complementation plasmid....

...CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via FACS, primer validation...article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized...CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations...deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given locus ...screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones... His lab studies the molecular genetics of blood cell development and disease. Learn more at his webpage...Kim JS. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 2010 Jan...

...with Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell Stem Cell . 17 (2), 221–232. PMID: ...in the same cell. This is called multiplexing , and increases the likelihood that any cell containing ...can increase efficiency. Synchronizing cells or modulating the cell cycle can help as HDR takes place during...expressing stable cell line. After applying your screening conditions to your transduced cell population, you...tracking of RNA in live cells, including orthogonal RNA/DNA labeling in the same cell. Figure 11: Overview...promiscuity is unique to bacterial cells and does not occur in mammalian cells. SHERLOCK , developed by the ...genomic loci in living human cells by an optimized CRISPR/CAS system. Cell . 155 (7), 1479–1491. PMID: ...

...prevent cell death. One of the cool things about amp is that it's exclusively toxic to dividing cells. The... continuous cellular processes (e.g., protein synthesis), while amp only affects the cell wall synthesis...Check icon" %} Spectinomycin Excellent No Just bacteria Not SHuffle cells {% icon icon_set="fontawesome...icon" %} Zeocin Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set..., penicillin, and is a beta-lactam that inhibits cell wall synthesis. The AmpR (amp resistance) gene produces...synthesis step during cell division. This is nifty because the recovery period after transformation (the dreaded...at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase prior to...

...transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However, if you ...(POI) to GFP to study the protein's behavior in cells, in solution or something in between, you are using...fluorescent protein (FP) could be forming dimers in cells. Why should you care? Three simple ways a dimeric...nanomolar Kd will exist primarily as a dimer in cells while a protein with a high micromolar or low millimolar...millimolar Kd is unlikely to form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple...might think EGFP is unlikely to form dimers in a cell. Unfortunately, things are not quite so simple. ...research situations, the relevant volume is that of the cell. For the physical behaviors associated with a molecule...

...numerous cell biology assays. However, there has been a pervasive view that most FPs in a cell will fold...fluorescent fusion proteins in cell biology."Current Protocols in Cell Biology (2005): 21-4. Pubmed PMID...Lee. "Fluorescent proteins: a cell biologist's user guide." Trends in cell biology 19.11 (2009): 649-655...Fluorescent proteins in cellular organelles: serious pitfalls and some solutions." DNA and cell biology 32.11 (...-fusions on quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent Protein...quantitative imaging experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable...dynamics of fluorescent protein fusions in live cells in real time. Previously, we have described a number...

... 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...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...be able to validate their findings in mammalian cells with both human and murine targets thus informing...

...on freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically... into a host cell (through a process called transformation), essentially giving the cell all the instructions...to dispose of the live cells. The alternative is to express the protein in a cell-free manner. This is ...electrophoresis to separate DNA or transforming cells with green fluorescent protein, these activities...transcription and translation. Unfortunately, having live cells in a classroom presents equipment needs, such as...operating the rest of the car as well. Similarly, cell free expression systems contain all the parts needed...handle a living, replicating organism. Furthermore, cell free systems can be freeze-dried into pellets (FD-CF...

... where many different cell types, or even different phenotypes of the same cell type, are intermingled...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...Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Kanaan, N. M., Sellnow...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...co-receptors, and other mechanisms to gain entry into the cell (Dudek et al., 2018; Korneyenkov & Zamyatnin, 2021...

... 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...infection for efficient cell lysis and release of adenovirus from infected cells. J Virol, 70(4), 2296.... & Verma, I. (1998). Cellular immune response to adenoviral vector infected cells does not require de ...production and/or by genes present in the HEK293T cell line used (which have fragments of the AdV genome...achieves persistent transgene expression in certain cell types (Tatsis et al., 2007), potentially because...

...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...find optogeneic actuators and sensors at Addgene. Cell marking/selection: Expression constructs like plasmids...

...4,000 cell lines. Seventeen of these cell lines were used to investigate the susceptibility of cells from...additional cell line used in this study came from the Japanese Collection of Research Bioresources Cell Bank...that SARS-CoV, which entered human cells via hACE2, could not enter cells expressing murine ACE2 (Subbarao...repositories that distribute mouse strains, plasmids, cell lines, and much more have been critical for pandemic...from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV...accelerating research into this virus. One such example is cell lines. The American Type Culture Collection (ATCC...of this pathogen. The sharing and distribution of cell lines from repositories on opposite sides of the...

.... 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...flourescent proteins that can be inserted into a cell without disrupting its functions. Recently, Ravid...

...difficult-to-transfect cell types ranging from primary to embryonic stem cells. Viral integration of a... also generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...Viral applications Viruses work by infecting a host cell (the target of viral infection) and delivering a...to be anything you want delivered to your target cells. This delivered DNA/RNA can then be either permanently...permanently integrated into the genome of the target cell or transiently expressed. It’s important to note...This vector can express your construct in target cells transiently or constitutively depending on the virus...transcription factors, barcoded libraries to trace cell lineage, and so much more! Libraries allow you to...

...fluorescence-activated cell sorting (FACS) helps researchers sift through thousands of cells to identify cells exhibiting...target single cells. Now, Francois St-Pierre’s lab developed a new form of FACS-based cell screening, Single-cell...individual cells with unique spatiotemporal phenotypes from large heterogeneous cultures.  HeLa cells expressed... conformational changes in a protein.    Cells expressing an RFP reporter plasmid for recombinase...is active in vitro as well as in human and plant cells. Casɸ from Biggiephage can eliminate competing... Single-cell Phenotypic Observation and Tagging with Light (SPOTlight) as a way to isolate individual ...of keratin with mGold (top) and mVenus (bottom). Cells were imaged every 30 seconds for 3 hours. Image ...

...cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer, Inflammation and Immunity and...recent findings in basic immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new...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...therapeutics such as  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in ...combination of the CRISPR/Cas9 methodology with T cell receptor engineering (Ref 5)  as described by Blue...

... on the method used to introduce the gRNAs into cells. Most CRISPR pooled libraries have been created ...the chance of transducing a only a single gRNA per cell. The viral DNA containing the gRNA then integrates...next-generation sequencing of the genomic DNA. However, some cell types are poorly transduced by lentivirus, and thus...CRISPR libraries was not possible. Primary murine T cells cannot be transduced by lentivirus and have been...library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express Cas9 and screen...CRISPR pooled library can be used for screening of cells that are refractory to lentiviral transduction, ..., now enabling forward genetic screens in cells with few genetic tools. Find the mouse CRISPR knockout...

...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)....timers, researchers can look at a single image of a cell and understand how protein localization changes ...was a tetramer with a propensity to aggregate in cells (Tsuboi et al.), limiting its use. Nonetheless, ...versatile timer could be used in vitro, in mammalian cells, in C. elegans, and in Xenopus embryos. Of course...

...packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids...precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV lysis buffer and freeze...bath to release the virus particles from the cells. Cell lysates are then treated with benzonase immediately...five days after transfection, the AAV-containing cells and media are harvested and purified. In total, ...not counting the time needed to expand the HEK293 cells. Check out the Addgene AAV Production protocol for... strains, like NEB Stable. NEB Stable competent cells lack RecA, a protein that aids in the homologous... AAV. I skip the PEG precipitation of the HEK293 cell culture supernatant because it’s time consuming ...

...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/...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...released into the culture media, while others remain cell-associated (Vandenberghe et al., 2010). For certain...

... between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j.cell.2023.05.044. PMID...lentivirus and MMLV gag, to specific cell types. It can also make CAR-T cell therapy more accessible by simplifying... efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419...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 ...DIRECTED plasmids here! Strebinger, D., et al. (2023). Cell type-specific delivery by modular envelope design...

... 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...

... 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...

...produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells are fused with activated...activated B cells from the spleen of mice injected with the antigen of interest. The immortalized cells, called...distributing over 3,000 antibodies and associated hybridoma cell lines. The DSHB was created by the National Institute... individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing...

...help in the capture of other rare cell types, like stem cells or cells infected with malaria or other parasites...tumor markers to subsequently capture cancerous cells expressing low levels of tumor antigen. I asked ...looking at, magnetic isolation of circulating tumor cells, we found that the binding stability really mattered... in the pili that they use to anchor on to host cells. Addgene: Were there any major hurdles along the... donor for keeping going while we worked out the cell capture system.) Cancer and other applications Addgene...cancer? Howarth: The capture of circulating tumor cells (CTCs) is going to have a big impact on our understanding...polymerizes affibodies to enhance magnetic cancer cell capture. Fierer et al (Proc Natl Acad Sci U S A....

... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ...plasmid to generate a population of mutant cells. The mutant cells were treated with WNV at a dose and duration...regulate the ability of WNV to induce cell death in infected cells (SEL1L, UBE2J1, EMC3, EMC2, DERL2, UBE2G2...AID-tagging system to essential genes in human cells and mouse ES cells because it was difficult to fuse the AID...-to-study genes in hard-to-study cell types. Natsume T, et al. Cell Rep. 2016. PubMed PMID: 27052166 ...small GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...therefore varies with cellular location: it is both red and green through much of the cell, but turns more ...

... 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...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...is very amenable to genetic manipulation. Yeast cells are great model organism not only for the reasons...

...nucleotide variant lines in an isogenic cellular background. All cell lines and phenotypic datasets that result...CRISPR Guide CRISPR Protein Tagging Plasmids for Stem Cell Research Neurodegeneration Research Collection The...Plasmids The following plasmids can be used to create cell lines with endogenously-tagged gene variants or ...or to differentiate human iPSCs into other cell types, as noted in the purpose field: ID Plasmid Description...neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH. Neuron . 2021 Apr 7;109...

...manipulations on cells, and has potential use for a wide range of applications in cellular biology. Van ... of a microtubule-associated protein to regulate cell growth dynamics Article contributed by Klaus Wanisch...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...Van Haren J et al. Nature Cell Biology 2018 Pubmed PMID 29379139. The “Riboglow” platform for visualizing...used to visualize RNA localization in mammalian cells. These tools consist of cobalamin-binding riboswitches...proteins (FP) with improved tissue penetrance and live cell imaging capabilities. Upon developing a new imaging-based...

...Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with cell-based method ...Requires large cell population, senstive to time since cell fixing HTGTS Not reported Cell-based Identifies...method IDLV 1% Cell-based Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods... use single cell clones for in vitro CRISPR experiments. The likelihood that a single cell clone derived...picture of the impact of CRISPR editing in the model cell line or organism. Several options exist for unbiased...Osborn et al. 2016) and HTGTS (Frock et al. 2015) for cell-based experiments. These strategies can be used ...off-target editing events. Two of the most common cell-based methods are genome-wide, unbiased identification...

...mammalian cells. Here, we will cover how these structures are generated, why it’s important for cells to sense...., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat Rev Mol Cell Biol, 23: 286...performs almost all of its biological functions in our cells in the single strand form, but double stranded RNA...with bidirectional transcription. In an uninfected cell, dsRNA rarely circulates, but upon viral infection...Unlike dsRNA, DNA:RNA hybrids are abundant in normal cells and are arise from a ssRNA engaging one strand of...genes is constantly occurring throughout a single cell, so these nucleic acid hybrids are always around...at risk. Consequences of duplexed RNA Mammalian cells have multiple receptors in place to sense dsRNA ...

...and inside the cells and are thus typically used for distinguishing live from dead cells. The combination...say I am culturing HEK 293T cells in vitro. I transduced the cultured cells with a virus that encodes a...panel. Besides some background cell death constantly happening in cell culture, the transduction process... to some cell death. As a result, there is a high abundance of positively stained dead cells present and...When analyzing your cells using flow cytometry, you are typically measuring the presence or absence of...certain markers on the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence...Another way to label proteins is through treating cells with fluorophore-conjugated antibodies that attach...

...transfected into a packaging cell line, usually a derivative of 293T. Packaging cells are incubated and after...fine-tuning selection and expression in your target cell line, researchers often find themselves faced with...impact the transfection efficiency of the packaging cell line and thereby affect the titer of the virus. ...layer flask will require some initial optimization; cell number, media volume, and plasmid concentrations...larger vessel to the 10 cm2 dish and adjusting the cell number, plasmid concentration, and experimental ... consideration when titering a lentivirus is the cell line that is used for titering. Typically researchers...researchers will choose a relatively permissive cell line for titering in order to obtain the best titer possible...

... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...delivery across species. Cell, 184(19), 4919-4938.e22. https://doi.org/10.1016/j.cell.2021.08.028 Vormstein-Schneider...capsid, (b) cargo, and (c) delivery method determine cell type- or region-specific transgene expression. (...include variants with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons...sequences, denoted by triangles] facilitate tissue or cell type specificity. An on/off genetic switch can be...

...Drosophila cells. N terminal tagging in Drosophila cells 3.2 MB C terminal tagging in Drosophila cells 3.3 MB...following article: Natsume, et al. Cell Reports 2016 Förstemann Drosophila Cell Tagging System The Förstemann... Allen Institute for Cell Science Plasmid Collection The Allen Institute for Cell Science has produced...tagged, human, induced pluripotent stem cell (hiPSC) lines. These cell lines were created using CRISPR and...tagging transcription factors with FLAG in human cell lines. To repeat the tagging in your system, use... from the Doyon Lab’s publication: Dalvai et al. Cell Rep. 2015 . Alternatively, cDNAs can be cloned directly... CRISPR tagging technique for use in Drosophila cells that uses PCR to generate both an expression cassette...