We narrowed to 490 results for: cel.2

...structure-based framework engineering. Methods, 34(2), 184–199. PubMed PMID: 15312672. Inbar, D., Hochman...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat the issues... detects the widely used HA epitope in diverse cellular environments. They called this probe the HA frankenbody...previously proven to fold and function in living cells. To make these scaffolds specific to different targets...the tagged protein upon probe binding in living cells.  In their proof of concept experiment the Stasevich...mCherry, 4xHA tagged histone protein (H2B) in U2OS cells. They then showed that the HA frankenbody probe ...

...MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: ... there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical...understand the connection between molecular- and cellular-level events and behavior. It’s a problem that...advantage for observing events taking place inside cells that are on the move, such as T lymphocytes. Griesbeck...-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish TnC variant which had...cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making this really good,” Griesbeck...FRET-based sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc...

... & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414 (2010). Cleveland...negative bacteria is due to differences in cell wall composition. Cell wall structure Gram positive bacteria...complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it dehydrates...

... and whether I would be able to carry out a yeast-2-hybrid library screen; instead I thought they would...would continue on with my academic dream of studying cell division. However, after 6 years in the business...Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial ...information on ELISA troubleshooting, ELISA kits and cell death assays. You can find Seán on twitter @Macfhearraigh...

...created for the Brunello library is shown in Figure 2. You can use a small PCR product because the plasmids... Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation...DNA. This bias can be counteracted by growing the cells on solid plates, where each transformant forms its...own colony, uninfluenced by the growth of other cells. For libraries where the plasmids are all very similar...

...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬👩‍...four main categories: anatomy and the human body, cellular biology, medical specialities, and general items...

... master's degree prepared them for their career.  2. Are you looking for career advancement and growth... or so I thought. So I entered a molecular and cellular biology PhD program in 2011, which I left four...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time... to mold it all into a full laboratory. It is priceless to see our “toys” gaining life and working as ...

...much has been done with CRISPR technology in just 2 years and excitement about how much more there is ...alone were worth the trip. Dr. Dana Carroll's excellent keynote address was the first of 19 talks given...

...available to the community. We now offer AAV9 for 2 more constructs from Christopher Harvey’s lab. These... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...

...of translation 2-10 ug/mL G418/Geneticin neo HeLa, NIH3T3, CHO, 293HEK, Jurkat T cells Blocks polypeptide...transfected cells may be considered an entirely new cell line from that of the original parental cells. Positive...cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are...bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells is fairly...expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is expressed from the...components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr...mammalian cells To achieve stable transfection, there should be a selective pressure to force cells to incorporate...

...differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced with the TF pooled...human embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed...transduced human embryonic stem cells with the viral TF library, with each cell receiving only 1 TF expression...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA sequencing... 206 cells per TF ORF. From this large sequencing endeavor, a map of possible TF-induced cellular states... clusters of individual cells along with color coded nominated reference cell types.  (Adapted from Joung...delivered to each cell, future applications could deliver multiple TFs to each cell, allowing for combinatorial...

...enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation protocol...the producer cells and purification protocols often describe a process that begins with cell lysis to release...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...

...is the most charged, with 5 negatively charged and 2 positively charged amino acids. FLAG tag has been .... This tag is not suitable for use in apoptotic cells as Caspase3/7 both cleave after the DVPD sequence...fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry, or immunofluorescence...

...yourself greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter...would rather fund excellent people with mediocre ideas than mediocre people with excellent ideas. I am lucky...

...incorporation of biosensors, protein evolution lineages, 2-photon spectra, more elaborate spectrum analysis tools...  There are many online guides – including an excellent series of posts by Joachim Goedhart on the Addgene...Associate at Harvard Medical School. He manages the Cell Biology Microscopy Facility, specializing in advanced...

...create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes within...isolating the B cells from an immunized animal, those B cells are fused to myeloma cells at a 1:1 ratio...fusion between an antibody-producing B cell and a myeloma cell (National Research Council (US) Committee...immortal cell population that will continue to produce the specific antibody that the original B cell produced... selects against unfused myeloma cells and antibody-producing B cells that cannot replicate, meaning that...monoclonal antibodies are derived from a single B cell clone. One common way of producing monoclonal antibodies...with BioRender.com.   The antibody-producing B cells that are used to make hybridomas come from animals...

...library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express Cas9 and...genome-wide interrogation of the genes required for cellular processes. For a successful screen, it is necessary... 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...CRISPR pooled library can be used for screening of cells that are refractory to lentiviral transduction, ...

... therapy.” Journal of cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. ...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...CRISPR in adult stem cells has huge potential, because the genomes of adult stem cells in organoid cultures...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank...homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (8).   The difference...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis....is a big advantage over induced pluripotent stem cells, which need to go through a phase of crisis where...

...improve the brightness of previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! ...have become indispensable tools for molecular and cell biologists over the last two decades, but their ...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...quantum yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent...three luminescent proteins can be used together in cells. As Nano-lanterns are not subject to photobleaching...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...

...to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant...cryopreserved hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of...of interest with a myeloma cell. This RNA was then used to generate cDNA, which  was used as a template...of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the mAb...hybridoma stocks, without having to grow up the cells. This RNA could then be used to generate functional...immunofluorescence-immunocytochemistry (IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target... could be screened in a high-throughput manner. Cells were labeled with both a hybridoma generated mAb...

...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...path.   Yeast as a model organism Yeast are single cell, eukaryotic organisms formally classified as fungi...and S. cerevisiae (budding). Yeast make for an excellent model organism for a few reasons: Affordable...similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number ...protein bang for your media buck - it grows at higher cell densities than most of its counterparts, yielding...oxidative reactions. They are found in all eukaryotic cells and peroxisomes are where methanol metabolism occurs...in methylotrophic organisms. K. phaffii is an excellent model for peroxisome studies since their peroxisomes...

...labeling of different cell types within a sample to expected labeling (Figure 2). The panel of antibodies...the antibody in the same tube.     Figure 2: Schematic showing the use of an orthogonal approach...levels in your cell lines, you can consider a cell treatment approach. If there is a cell treatment (e....select a candidate cell line (e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen...the parental cell line, but not in the iso-genic knockout control. This can involve cell surface labeling...selectivity for cell surface labeling may become less selective if used in intracellular labeling because...because the intracellular compartment may contain cross-reacting antigens not present on the cell surface....

...retransduce a model organism such as mice, e.g. first at 2 months, then at 4 months? Or do antibodies become ...co-delivering different AAV particles into the same cells in vivo? A: This will be highly dependent on vector...particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver, we recommend...worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently recommend...and to all our participants for their time and excellent questions.    Additional Resources on the Addgene...

...axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses can be injected...they either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...particles require receptor-mediated entry into the cell. This means that not all neurons and pathways will...chemogenetic vector directly into the region where the cell bodies of the projection neurons reside.   Impact...

...delivering an AAV construct expressing channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused...and chemically excited a class of enteroendocrine cells within the intestines known as neuropods. They observed...

...extraction protocol for more details.   Figure 2: An uncut plasmid is expected to form 3 bands: nicked...efficient genome editing in Escherichia coli. Microb Cell Fact 19. https://doi.org/10.1186/s12934-020-01321...

...containing complementary polynucleotide chains. J Virol 9(2): 394-396. Lecomte E, Tournaire B, Cogné B, Dupont... small amount of spurious DNA from the packaging cell line, helper and packaging plasmids. We decided ...

...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...Non-Dividing Cells Transient Gene Expression Low Tropism for Mouse Cells and Hematopoietic Cells Low Packaging...fuses with the target cell’s membrane, allowing the viral particle access into the cell. The viral envelope... of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...viruses can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo...wide range of cell types that are typically ‘difficult to transfect’, such as primary cells. They can also... them to easily deliver their “cargo” to target cells and organisms, and when that cargo is designed to...

... would release them into the cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids...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... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such as...While these molecules are clearly associated with cellular damage and dysfunction, it is unclear what the...

...Gene and Cell-Based Therapies in the New Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. ...dividing cells only, so is not suitable for infection of quiescent cells or infrequently cycling cells but ...non-dividing cells, making it conducive to targeting all cell types. Altogether, AAV has many excellent features...DNA copies of their genome upon infecting a host cell. This viral DNA (also called proviral DNA) is then...and permanently integrate a genetic cargo into a cell. These viruses have a cargo capacity of ~ 8 kb, ...needs, and can transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus...but is fine for experiments with immortalized cell line cultures. Lentivirus Lentivirus is a type of retrovirus...

... Figure 2: Isotype switching requires DNA recombination of the antibody gene locus. B cells start by ...the surface of basophils and mast cells, two types of white blood cells involved in allergic reactions. ... is important because it determines what immune cells and molecules are recruited by the antibody to help...each of these functions: Recruitment of immune cells that express receptors that recognize the Fc portion...which can initiate a cascade that helps attack extracellular pathogens.  Transportation of antibodies to ...special receptors that transport antibodies through cells and into different body compartments, such as into... during a primary immune response because all B cells begin by expressing IgM. IgM has a pentamer structure...

... vaccines, including 1) cellular responses in addition to antibody response 2) very high immunogenicity...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...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV .... 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...

... the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propagate per cell. 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. Similarly, the specific ORI elements included within...

...closely mimicking the future application. In figure 2 we provide an example of the comparison of the practical...dodge cellular variation observed in mammalian cells is to tag an endogenous gene. By imaging cells (or ...mammalian cells within the dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct...intended application. The three key properties for live cell imaging that will be treated in this series of posts...Determining practical brightness When a dish of mammalian cells is transiently transfected with a plasmid containing...allows for a precise determination of brightness in cells, under realistic experimental conditions. By determining...al (2017) and Heppert et al (2016). In mammalian cells this would be possible with CRISPR/Cas-based genome...

...acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes... including live-cell imaging, single-molecule tracking, immunolabeling of fixed cells, Western blotting...possibilities in both cell engineering and gene therapy.  Xiaoshu X, et al. Molecular Cell. 2021. https://doi.org...Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized genetic screening technique ...Anti-FLAG Frankenbodies for modular imaging of cellular targets by: Mike Lacy Adding to their set of anti-HA...well-folded scFv scaffold to improve its stability in cells, and use it in a variety of applications. This set...frankenbodies to amplify the signal.  Liu et al. Genes Cells. 2021. https://doi.org/10.1111/gtc.12893, Zhao et...

...aspects of the work you presented may also be unclear. 2. Make the audience your primary focus When writing...as an opportunity to practice communicating.  Excellent scientists learn from advisers and other mentors...help graduate students in all STEM fields become excellent science communicators. To empower graduate students...

...higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive...This novel tool is practical in cell and organ culture, as well as cell lineage studies. Eisenhoffer and...CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this system, cells express...fast-splicing split intein and co-infected into the cell. Intein splitting removes any exogenous sequences...split into two plasmids and co-transfected into cells for reconstitution. Image from Levy et al., 2020...red-light inducible optogenetic tool for precision in cell lineage tracing Article contributed by Erin Sanders...fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene that recombine...

...about colony PCR in this blog post.   Figure 2: The steps of colony PCR.   Sanger sequencing Sanger...activity. These cells, presumably the cell you want, cannot break down X-gal and are white. Cells that don’t...killing off a portion of the cells like in a selection. Instead, all cells survive and you will need to... the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using...using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic... plates containing X-gal allows you to identify cells that took up the vector and distinguish between ...

...PMID: 20543841. PubMed Central PMCID: PMC2920597. 2. Nathans, Daniel. "Puromycin inhibition of protein...proteins in cells. Homeostatic regulation of protein abundance is essential for normal cell function, and...one particular protein in neurons or in any other cell type. However, we cannot distinguish new from old...facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed from...techniques to follow translation in living single cells. However, before CRISPR-Cas9 these techniques were...interests, suspected to interact, are added to a cellular sample. Both antibodies have a short antibody ... turnover of specific endogenous proteins in any cell type. Exciting! Many thanks to our guest blogger...

...available in the adenovirus section below!   Fig. 2 - Viral vector components of AAV Adenovirus Adenoviruses...genetic cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA, or something...perform very similar functions to infiltrate the host cell and deposit viral cargo. Gene expression While AAV...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...altered tropism of AAV to target different tissue or cell types. AAV also requires some back up from adenovirus...promoter that you wish to deliver to your target cells. In adenovirus, the ITRs serve as a type of origin...deficient. This is typically supplied by the production cell line (HEK-293) during production. Fun fact: There...

.... PubMed PMID: 6276558. 2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of...transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination. What...downstream of promoters that are active only in certain cell or tissue types, during certain stages of development... recombinase can be expressed only in specified cells or at specified times. Combining this with some ...has been used to successfully introduce Cre into cells of interest. What if I need two separate recombination...

...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(...advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing...tRNAs are among the most abundant molecules in a cell, this system is clearly also robust enough to ensure...54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. PubMed.  Find the plasmids from this...

...pluripotent stem cells differing exclusively at two early onset Parkinson Point mutations. Cell, 146(2), 318–331...extraction solution, runs the protocol, then uses around 2–4 µl in each 50 µl PCR reaction. The gDNA extracted...find both single-allelic and bi-allelic cells. Single-allelic cells usually make up the majority in culture...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....Double-Strand Break Repair in Mammalian Cells. Molecular and Cellular Biology, 18(1), 93–101. https://doi....genome editing specificity. Cell, 154(6), 1380–1389. https://doi.org/10.1016/j.cell.2013.08.021  Ran, F. ...CAS-Mediated Genome Engineering. Cell, 153(4), 910–918. https://doi.org/10.1016/j.cell.2013.04.025  Yang, H....

...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Screen identifies... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Rapid and phosphoinositol-dependent... Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. 1998 Nov 25. 95(5):625-36. An architectural role..., Ensminger AW, Fox AH, Chess A, Lawrence JB Mol Cell. 2009 Mar 27;33(6):717-26. Reporter-nanobody fusions...

...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally... 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...

..., but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple...bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and...nicked plasmid together long enough to get it into cells and let them do the repair work. The key is T4 DNA...

...-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware (right...quantitatively evaluating antibody staining, or counting cells are now routine, open-source software projects that...portable device for studying plankton using flow cells, has become an established alternative to high-priced... and blueprints has the potential not only to accelerate scientific progress, but also to enable science...

... scientific communication ecosystem Figure 2: The scientific ecosystem, enriched by open access...from peer-reviewed publishing in a journal.  Accelerating scientific communication Preprints have been...Figure 1: Stand aside, CERN — it's a new age of accelerating science! Photo courtesy of Henry Mühlpfordt,...Lacy was also employed by the American Society for Cell Biology in a position working with preprints in ...