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Showing: 151 - 180 of 820 results

  1. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...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 ...

  2. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ...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 ...

  3. Hot Plasmids - August 2020

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    Blog Post
    ...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...

  4. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

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    Blog Post
    ...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 ...

  5. 28 Hot Plasmid Technologies from 2015

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    Blog Post
    ...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...

  6. Viral Vectors 101: What is a Virus (and a Viral Vector)?

    Type
    Blog Post
    ...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...

  7. FlipGFP, a novel fluorescence protease reporter to study apoptosis

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    Blog Post
    ...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 ...

  8. Plasmids 101: Screens vs. Selections

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    Blog Post
    ...culture cells in the drug of interest at a lethal dose for non-manipulated cells and then assay cells which...selection, you might put cells into a drug-containing media in which only cells which can tolerate the ...considering either a cellular screen or a selection. A selection is a process in which only cells with the trait... – 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...

  9. Plasmids 101: Yeast Vectors

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    Blog Post
    ...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. Similarly, the specific ORI elements included within...plasmid) allows for 50+ copies to stably propagate per cell. The copy number of these vectors can also be controlled...

  10. Hot Plasmids February 2024

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    Blog Post
    ...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...

  11. Your Lentiviral Plasmid FAQs Answered

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    Blog Post
    ...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...

  12. Cell Migration Consortium Plasmids

    Type
    Collection
    ... Depositor Collections Cell Migration Consortium (CMC) Plasmids Cell Migration Consortium (CMC) Plasmids...collaboration between the Cell Migration Consortium and Addgene. Member laboratories of the Cell Migration Consortium...This resource is a collaboration between the Cell Migration Consortium (CMC) and Addgene, which highlights...Consortium have deposited plasmids useful for cell migration research at Addgene. CMC investigators who have...

  13. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

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    Blog Post
    ...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 ...

  14. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ...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...

  15. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    ...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...

  16. dTAG - You're it!

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    Blog Post
    ...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 ...

  17. Plasmids 101: Blue-white Screening

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    Blog Post
    ...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....

  18. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

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    Blog Post
    ...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...

  19. Plasmids 101: Choosing an Antibiotic Resistance Gene

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    Blog Post
    ...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...

  20. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

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    Blog Post
    ...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...

  21. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

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    Blog Post
    ...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...

  22. Supporting Rare Disease Research with the 2020 BeHEARD Awards

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    Blog Post
    ... 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...

  23. Creating accessible biology activities in schools with BioBits

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    Blog Post
    ...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...

  24. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ... 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...

  25. Viral Vectors 101: AAV Serotypes and Tissue Tropism

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    Blog Post
    ...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 ...-associated viruses. Cell, S0092-8674(25)00692-0. https://doi.org/10.1016/j.cell.2025.06.026 Halbert, ... Therapy: An Overview. Cells, 12(5), 785. https://doi.org/10.3390/cells12050785 Kaludov, N., Brown, K....tissue tropism, allowing them to target particular cell types. Figure 1: Diagram of AAV components...key that lets AAVs “unlock” and infect specific cells. This protein shell packages the vector’s genetic...multiple coreceptors, which may be present on different cell types and sometimes vary between target species ...

  26. An Introduction to Adenovirus

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    Blog Post
    ... 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...

  27. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

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    Blog Post
    ...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...

  28. Reagent Repositories Are Speeding up Science During the Pandemic

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    ...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...

  29. Hot Plasmids - May 2022

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    Blog Post
    .... 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...

  30. Viral Vectors 101: Viral Applications

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    Blog Post
    ...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...
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