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We narrowed to 1,029 results for: ELL

Showing: 401 - 440 of 1029 results

  1. Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

    Type
    Blog Post
    ...identify genes essential for cell viability in cancer and pluripotent stem cells. In a melanoma model, they...high representation of library within cells and aimed for 300 cells per construct, but in some biological...were able to produce a very diverse population of cells within a lab dish in a matter of a few days. Their...pooled libraries include enrichment against kinases, cell cycle proteins, and more.  “In the past, genome ... delivered via lentiviral infection into target cells that have previously been stably transfected for...conduct systematic genetic screens in mammalian cells,” David Sabatini, a senior author of one of the ...understand the function of both protein-coding genes as well as non-coding genetic elements.” These lentiviral...

  2. Deep Dive: qPCR

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    Blog Post
    ...bubbles in wells, variations in volume, or machine surge errors and allow for well-to-well normalization...reported to work well in the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table...Bustin SA; Benes V; Garson JA; Hellemans J; Huggett J; Kubista M; Mueller R; Nolan T; Pfaffl MW; Shipley... that your reference genes are suitable for your cell lines and/or tissue types.  Please do note my oh-so-subtle...reference genes to save on plate space. For many cell lines and tissue types, appropriate reference genes...at determining the best options for a particular cell or tissue type (such as here, here, or here).  If...select the appropriate reference genes - and it is well worth your time to do one (Biassoni, 2014). At the...

  3. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem Cell 15:27–30 . https://...protein, in order to mitigate the ability of the cell to use an alternative ATG downstream of the annotated...process, and usually involves the need for single cell cloning and subsequent screening for successful ...standard requires not one but two rounds of single cell cloning – as a control, one should revert the edit...passenger variant that came along with the single cell clone (although this is rarely done). When targeting...can affect the accuracy of a predictive score, as well as dynamic aspects of the target (e.g. accessibility...levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...

  4. Plasmids 101: Repressible Promoters

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    Blog Post
    ...functions well in mammalian cells, and can be used in the repressible manner described above, as well as the...bacterial origins, Tet systems function well in mammalian cells, and TRE-containing promoters can be used... by ~3,300-fold in Drosophila cells and ~24,000-fold in human HeLa cells as compared to basal expression...be switched from a default ON to an OFF state, as well as repressible binary systems. In recent years, ...used in the repressible manner described above, as well as the inducible manner detailed in our previous...While originally developed for use in mammalian cells, this system has also been implemented in a variety...transgene permits directed expression of that gene in cells that also express GAL4. Usage of this system revolutionized...

  5. Antibodies 101: The Basics of Western Blotting

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    Blog Post
    ..., transfected it into your target cells, now what? How can you tell if the protein you are so keen to ...readout for subcellular fractionations, a procedure that isolates proteins from different cellular compartments...to study is expressing in your cells? Immunoblotting or simply the western blot, or western, is one of ...specific protein within the complex mix derived from cells or tissues. Westerns are useful not only to detect...protein levels relative to standards, detect the cellular location of proteins, and can be a readout for...immobilized onto a membrane, usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step...step called protein transfer. While both nitrocellulose and PVDF are commonly used, PVDF tends to be slightly...

  6. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

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    Blog Post
    ...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... by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics with light by modifying EB1. EB1 normally...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... opens novel ways in manipulating and studying cellular dynamics. The photoinducible control of protein...Van Haren J et al. Nature Cell Biology 2018 Pubmed PMID 29379139. The “Riboglow” platform for visualizing...

  7. 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 ...Viruses are simple: a genome packaged in a protein shell (Taylor, 2014). They’re so simple that we can’t ...While all viruses have a genome inside a protein shell known as a capsid, some also have a lipid bilayer...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...

  8. Uncovering Ancestral Bias in CRISPR Research

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    Blog Post
    ...guides, short pieces of RNA that bind to a cell’s DNA, telling the Cas machinery where to cut.      ...Biomedical research has a long and well-documented history of racial bias, which still impacts and influences...ancestry and germline sequence variation in cancer cell lines took them down unexpected paths. Though this...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of...to see if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American...or South Asian) used in the screens based on the cells’ germline genetic variations. They then wanted to...with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either...

  9. Tips for Screening with Yeast Two Hybrid Systems

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    Blog Post
    ...entire proteome of a particular cell type, or a subset based on a cellular compartment or growth stage)....strain for screening), or arrayed on a 96 well plate where each well contains a defined prey fusion.  The ...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...extensively to screen for molecular interactions in the cell, including protein-protein, protein-DNA and protein-RNA...field of eukaryotic transcriptional activation and cell biology. During this period, proteins were successfully...The reporter gene then allows detection of those cells where binding between the proteins of interest has...alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the problem is that your proteins are not post-translationally...

  10. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...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...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, ...

  11. Ghanaian Researchers to Receive Plasmid Awards from Addgene and Seeding Labs

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    Blog Post
    ...Addgene to advance their research. Louis Bengyella Dr. Bengyella’s work has focused on elucidating the molecular...his future work. Dr. Bengyella would like to use the expertise he’s developed as well as CRISPR-based gene...chimeric cellulases that enable E. coli and Citrobacter freundii to generate sugars from cellulosic biomass...our first two plasmid grant awardees: Drs Louis Bengyella and Kwabena O. Duedu, both from the University...in Ghana. Read on to learn more about doctors Bengyella and Duedu and how they plan to use plasmids from... microbial pathogens. This work has given Dr. Bengwella expertise in using bioinformatic and proteomic...development of uterine tumors. According to Dr. Bengyella, this class of tumors is “a major public health...

  12. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

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    Blog Post
    ... are filled with early mitotic cells as well as necrotic or dead cells (Jiang and White-Cooper, 2003)....a key role in proper limb and digit formation as well as neural development. Specifically, SHH is a morphogen...Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene called "...the fly actomyosin cytoskeleton that regulates cellular contraction. This network is composed of actin...interact with each other to generate forces that cause cells and muscles to contract depending on the context...context. Contraction is critical for processes such as cell movement/motility, development, cytokinesis, and...scientists who discovered comr thought the sperm cells in mutant flies looked like a “whole bunch of cookie...

  13. Viral Production at Addgene

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    Blog Post
    ... preparations are added to cells in culture. Three to five days later, cell cultures are inspected for...a new vial of early passage cells. Approximately two weeks post-thaw, cell culture supernatant is tested...section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445...preparations ("-LV” catalog items) are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging...packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first...(PEG) followed by centrifugation. Precipitated pellets containing viral particles are then resuspended...

  14. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...efficient prime editors. Cell, 186(18), 3983-4002.e26. https://doi.org/10.1016/j.cell.2023.07.039 Lin, Q.,...correct only a minority of those variants in most cell types. But prime editing, a CRISPR technique developed...unedited DNA. The heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing...than prime editing. But for positions that aren’t well positioned within the editing window, prime editing...editing demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment can all impose ...

  15. Screening for Successful Genome Editing with Digital PCR

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    Blog Post
    ...analyzed gDNA from a few thousand cells from single wells of a 96-well plate without prior quantification...was contributed by Scott Findlay, a Postdoctoral Fellow at the University of Alberta. If you’re like many...physically isolated partitions (such as droplets or wells on a chip), such that most partitions will contain...are many thousands of partitions, statistical modelling can be used to determine the actual number of ...and reverse primer to amplify the target locus, as well as a reference probe designed to bind at an unedited...to quantify rates of mutation in a bulk or single cell-derived genomic DNA sample as part of a precision... that they are easily able to distinguish single-cell derived samples with mono-allelic mutations versus...

  16. New and Upcoming Viral Vectors - May 2020

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    Blog Post
    ...Deisseroth 83895 AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics continues...Cepko 83898 AAVrg pAAV-mDlx-ChR2-mCherry-Fishell-3 Gordon Fishell   Chemogenetics Chemogenetics offers...pAAV-hDlx-GiDREADD-dTomato-Fishell-5 83897 AAVrg pAAV-hDlx-GqDREADD-dTomato-Fishell-4 Controls Plasmid...pAAV-mDlx-GFP-Fishell-1 Biosensors Plasmid Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2 ... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...many of our control plasmids in new serotypes, as well as a new Cre dependent GFP control plasmid for expression...offers noninvasive methods for manipulating cell activity, particularly in the brain, and holds promise in...

  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...pUC18 and pUC19 Let’s begin at the beginning. The well-characterized bacterial lac operon contains a gene...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 ...

  18. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

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    Blog Post
    ...proteins enable the observation of proteins in living cells and organisms. Both components of the chimera are...However, these may not reflect the situation in cells. Therefore, the localization of critical fusions...currently the standard for assessing oligomerization in cells. In addition to measuring stickiness, several researchers...fluorescent proteins that behave as monomers in mammalian cells include: mTurquoise2, mEGFP, mNeonGreen, mScarlet...figure 1). In fact, for several FRET biosensors (yellow cameleon 3.60, EPAC, and Galphai), the C-terminus...of the fluorescent protein into the POI can work well since the N- and C-terminal residues of the fluorescent...protein to the existing pool of protein within a cell and may lead to over-expression artifacts. This ...

  19. Hot Plasmids: Summer 2024

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    Blog Post
    ...epigenome editing. Cell, 184(9), 2503–2519.e17. doi: https://doi.org/10.1016/j.cell.2021.03.025.    Recombinant...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...Trimmer’s lab (Figure 5) as well as antibodies for identifying other cell types in the nervous system!..., particle orientation distribution and Fourier-shell correlation curve (D), and the reconstructed 3D ...particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs contain... will only be transiently present in transduced cells, limiting off-target editing. Cargo size limits ...site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime ...

  20. Overcoming the Challenges of Lentiviral Production

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    Blog Post
    ...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...

  21. BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development

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    Blog Post
    ... stem cells. The lab will use plasmids from Addgene to construct NFIX expression vectors as well as reporters...the physiological and pathological roles of human cell glycans. Accordingly, she joined efforts with the...aspect of immunology, participating in events such as cell development, recognition and migration, the immune... is not surprising. However, the molecular and cellular mechanisms behind the immune defects in CDG patients...plasmids to immortalize CDG patient-derived immune cell lines and use these lines to study how immune processes...and other pathological conditions. References: Monticelli, M., Ferro, T., Jaeken, J., dos Reis Ferreira...McMurray is an Associate Professor in the Department of Cell and Developmental Biology at the University of Colorado...

  22. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

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    Blog Post
    ... expressed in cells via plasmids or by the creation of stable cell lines and these cells are then grown... complex. Not performed in a native cell context. For AP-MS, cells are lysed before the bait protein is...and interacting proteins in mammalian cells. The Journal of cell biology. PubMed PMID: 22412018. PubMed... it allows for the validation of a binary PPI as well as the identification of additional interacting ...against PPIs that aren't stable outside the native cell context. The Split-BioID method Enter Split-BioID...protein interactions. However, FRET generally only tells you if two proteins interact and cannot be used ...tagging of proteins in both BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein...

  23. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    Blog Post
    ... require minimal cellular perturbation. Also, this method requires live, intact cells; the sequencing ...Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...mutations, and environmental conditions drive certain cellular behaviors, but little is known about the order... may be used to better understand and engineer cellular processes that are influenced by temporally ordered... one final state, then the state of the RSM will tell you precisely what inputs were added to the system... our paper (1), we allowed individual bacterial cells to form colonies on a plate and then we performed..., but more population-wide (as opposed to single-cell) approach is to interrogate state with qPCR. The...

  24. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

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    Blog Post
    ...metabolite in a cell. The only way to do that at scale right now is to grow up a bunch of cells, sacrifice ...capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks Foundation...transistor-like digital genetic switches that enable cellular computing. These logic gates built from transcriptors... Collection along with a BIOFAB kit comprised of well-characterized bacterial transcription and translation...as an operating system for engineering an entire cell. Typically when people use plasmids, they are trying... you could imagine engineering the entirety of a cell. That's a big long-term technical challenge. We ...That's central to everything that goes on inside a cell. Until we get a handle on precise, forward engineering...

  25. Plasmids 101: Positive and Negative Selection for Plasmid Cloning

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    Blog Post
    ... and zap the mixture of DNA into cells. Unfortunately, not every cell successfully takes up plasmid DNA...selection: Cells that have gained a specific gene survive In positive selection, only cells that contain...cassette, you would plate the cells on kanamycin-containing agar. Only cells carrying plasmids that contain...Therefore, your cloning strategy needs to identify cells containing the plasmid construct you’re seeking....resistance marker. After transformation, plate the cells on medium containing the corresponding antibiotic...these cases, antibiotic selection will select for cells that have been transformed with the plasmid backbone...plasmids to complement auxotrophy. Negative selection: Cells that have lost a specific gene survive Unlike positive...

  26. Understanding GPCR Signaling Using TRUPATH

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    Blog Post
    ... to environmental conditions, cells must be able to detect extracellular stimuli. One way they do so is...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...play many roles in signaling processes including smell, taste, sight, inflammation, and neurotransmission...complex converts a signal received by a GPCR on the cell’s surface into action that can result in the initiation...number of signaling cascades, inducing different cellular responses. One of the biggest advantages of such...protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing the GPCR... and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the luminescent enzyme substrate...

  27. AAVs in Retinal Gene Therapy

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    Blog Post
    ...expression in non-dividing cells because they remain in episomal form in the cell’s nucleus. But since nothing...hope are many more clinical successes for gene (and cell) therapies. One may wonder why the first AAV-based... providing long-term gene expression in retinal cells - AAV - were becoming available. At the same time... lentivirus (LV) are known to transduce retinal cells, AAV quickly became the vector of choice for ocular...long term expression in dividing and non-dividing cells due to its (random) integration into the genome,...genome, and its large DNA packaging capacity is well suited for larger transgenes. Unfortunately it has limited...limited tropism in the retina, primarily infecting cells of the retinal pigmented epithelium (RPE). AAVs...

  28. Viral Vectors 101: The Retroviral Lifecycle

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    ...synthesized inside of what is called the “target cell” (or host cell) by the viral protein reverse transcriptase...reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and translation...1971). Noninfectious ARK mouse embryo cell lines in which each cell has the capacity to be activated to ...tools in molecular biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses...gamma-retroviruses have their own unusual sort of virulence as well — namely that they can cause cancer via insertional..., interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses are RNA-containing...separate set of instructions in the same producer cells as the virus. Because it is made at the same time...

  29. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

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    Blog Post
    ...choose cell lines that are highly transfectable.  The amounts of each plasmid transfected into cells are ...100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate format). However, since plasmid quality... polymerase gene or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes...capable of being replicated and transcribed in the cell when the appropriate viral proteins are provided... RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...on separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome. These...transcribed (and the viral mRNAs are translated) by cellular machinery. NP encapsidates the negative sense,...

  30. Cpf1: A New Tool for CRISPR Genome Editing

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    Blog Post
    ...new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was annotated in several genomes and...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid...GC-content. After sequencing and searching for cellular RNAs important for Cpf1 function, they found that...the PAM. With this information, they turned to a cell culture system to see if any Cpf1 nucleases would...would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-...

  31. Finding Your Science Policy Path

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    ...developed by Dr. Kelly Singel which provides an overview of all available science policy fellowships in federal...Steph Guerra, a AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously... a variety of science policy roles including fellowships, volunteer positions, and part-time opportunities...current work as a AAAS Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I...pursue regulatory approval for a new product or a well-endowed philanthropy who can shift the funding landscape...compiled by the AAAS Science and Technology Policy Fellowship Program to learn more about where science policy...policy? Many science policy training programs and fellowships have a strong focus on early career researchers...

  32. Neuronal labeling with Spaghetti Monster

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    .... FPs are generally bright, stable and well tolerated by cells. They can be used for protein localization...of neuronal sub-cellular structures GFP is limited in its ability to label subcellular structures in the...ability to interact with protein partners within the cell. However, antibodies can have low affinity for these...trigger protein aggregation which is toxic for the cells and affects the labeling of small structures like...team, an ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with...is a symbol of opposition to the teaching of intelligent design in public schools and was started in 2005...used to amplify the direct smFP signal within sub-cellular structures. For instance, smFPs have been used...

  33. Visualizing Genomic Loci with CRISPR-Sirius

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    ...The catalytically dead Cas9 protein (dCas9) is well known for its ability to bind DNA targets without... to date for visualizing genomic loci in living cells. Previous iterations of genomic loci visualization...labeling system to visualize chromosomal loci in human cells (Ma et al., 2015). To do so, they fused a different...different hairpins will produce a secondary color (yellow, cyan, or magenta) depending on the hairpin combination...visualization of sites on different chromosomes in living cells, the system requires high-copy chromosome-specific... aptamer system to visualize the gRNA in living cells. Broccoli aptamers are nucleic acid sequences that...production needed to deliver CRISPR-Sirius into cells. They named this guide sgRNA-Sirius-8XMS2 which ...

  34. 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...

  35. Hot Plasmids - January 2023

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    Blog Post
    ...mCherry for FACS sorting of the weaponized T-cells (CAR-T cells) - and then delivering them to a tumor - ...effectively as standard CAR-T cells, but it reduces the off-target effects caused by T cell migration.  CAR expression...Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids here!   Campbell, B.C., et al. Chemically...reducing the on-target off-tumor activity of CAR–T cells was recently devised by the Yingxiao Wang lab.  ...above can be made for expressing your favorite T-cell receptor, or perhaps any other tool that can benefit...  Wu Y, et. al. Control of the activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed... is that some antibodies may just not perform as well in one application as another, so it is always best...

  36. Hot Plasmids - February 2022

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    Blog Post
    ...-binding scFv onto a well-folded scFv scaffold to improve its stability in cells, and use it in a variety... 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...frankenbodies to amplify the signal.  Liu et al. Genes Cells. 2021. https://doi.org/10.1111/gtc.12893, Zhao et... Cas12a for multiplexed gene perturbation by: Gabrielle Clouse While many CRISPR-based genetic screening...

  37. Plasmids 101: Modular Cloning Applications and Kits

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    ...can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants...transport studies in HEK cells, recombinant protein purification in bacterial cells, and a two-hybrid protein...for synthetic biology applications in bacteria or cell-free systems. The approach is particularly useful...tags to your proteins, then the CIDAR Kit is an excellent option (Iverson et al., 2016). CIDAR offers a ...add-on kit and is compatible with E. coli and/or cell-free systems. Protein expression in yeast MoClo ...plant-specific parts you‘ll need, including sub-cellular localization signals, plant markers, and reporters...of expression and integration needed in mammalian cell types, and comes with a large library of common ...

  38. With an Eye Towards the Future, We Look Back at the March for Science

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    ...training, so of course I felt compelled to participate in one of the satellite Marches for Science (my local...currently a post-doctoral fellow at Vanderbilt University. She studies beta-cell function and failure in... duty to tell our science stories in a manner that encourages open discourse with our fellow community...also incredibly impressed with how well organized the march was, as well as the friendly atmosphere that ...constant vigilance and actions beyond marches, as well as allies outside the scientific community. Vanessa...responsibility to join the public conversation and to tell the stories behind our research.  That's why I marched...against legislators and because the march had been so well-publicized, we might encounter hostile anti-science...

  39. Choosing the B(right)est Fluorescent Protein: Photostability

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    Blog Post
    ...intensity evolves over time in living cells at a power used for live-cell imaging. To conclude, the most photostable...molecule will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins...experiment. The best fluorescent proteins for live cell imaging can be excited many times, thereby producing...these factors, environmental conditions such as cellular redox state and oxygen concentration may affect...protein is a key requirement for quantitative live-cell imaging, it is important to be able to quantify ...it is advisable to perform time-lapse imaging of cells producing the fluorescent protein of interest under... a substrate. These in vitro methods allow for a well-controlled environment, but they do not mimic the...

  40. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

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    ...Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar S, Davies BW, Ellington AD, Moran NA, Barrick...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen... is primarily a means of selection that targets cells in which homologous recombination has not occurred...ensures high editing efficiency; the only non-edited cells to survive have inactivating mutations in the Cas9... E. coli, the system was used successfully in Tatumella citrea, another species of Enterobacteriaceae,...one or more genes in E. coli. and in a mammalian cell line (Qi et al., 2013). Two important features of...other Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 ...
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