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We narrowed to 587 results for: cel.2;

Showing: 51 - 100 of 587 results

  1. Adenovirus Plasmids

    Type
    Collection
    ...recombining shuttle plasmids Vogelstein 16401 pAdEasy-2 Adenoviral For recombining shuttle plasmids; for use...transgene under CMV promoter Vogelstein 16407 pAdEasy 2-GFP beta-gal Shuttle Test plasmid that contains β-...target a broad range of dividing and non-dividing cell types with almost 100% efficiency. Unlike lentiviruses...recombination of adenovirus plasmids in either mammalian cells or microorganisms, including bacteria and yeast....can then be transfected into mammalian packaging cell lines to generate adenovirus particles. Read our...

  2. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...colony size. Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with ... CO 2 incubator Pipet controller Hazardous waste container Microscope Hemocytometer or other cell counter... is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which...transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell lines from expansion of...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...

  3. Four Factors that Differentiate the Stem Cell Field

    Type
    Blog Post
    ... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients...explained that it took 50 people nearly 2 years to create the first stem cell line that could be used in clinical...prevent stem cell lines from becoming the new HeLa cells?), genome engineering and stem cells, and the difficulty...opportunity to see Shinya Yamanaka celebrate the tenth anniversary of his landmark Cell paper showing that it was...generate induced pluripotent stem cells (iPSCs) capable of forming nearly all cell types in the body from otherwise... happening. Stem cells scientists have certainly not lost site of that. 3. Stem cell science is hard, ...otherwise terminally differentiated cells simply by expressing four proteins Oct4, Sox2, Klf4, and cMyc...

  4. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...surviving cells. Even in the absence of cell death, the cell media should be changed every 2–3 days to...Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing selection...into two 75 cm 2 flasks, etc. Pro-Tip This selection method results in a polyclonal cell population, meaning...Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...

  5. Antibody Plasmid Collection

    Type
    Collection
    ...phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas Vector system for expression...+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102. Joanna Bereta Sybody Generation Toolbox for...expressing these in bacteria, mammalian cells, yeast, plants, or insect cells. Benefits include consistency between... research purposes include animals or hybridoma cells, but are difficult to generate due to expense and...monoclonal antibodies (R-mAbs) expressed in mammalian cells. Monoclonal antibodies are derived from an individual...transiently transfected into mammalian tissue culture cells, the R-mAbs are secreted into the culture media ...

  6. Adeno-associated virus (AAV) Plasmids

    Type
    Collection
    ...should be handled at BSL-2. AAV can transduce both dividing and non-dividing cells with a low immune response...expressing Rep2 and Cap1 James M. Wilson 104963 pAAV2/2 AAV2 AAV packaging plasmid, expressing Rep2 and Cap2...e.g., pHelper) and specific packaging cell lines (e.g., HEK293 cells) during AAV production. Read our AAV...

  7. Zinc Finger Consortium: Zinc Finger Arrays

    Type
    Collection
    ...gene modification. Maeder ML et al. Mol Cell. . 2008. Jul 25. 31(2):294-301. PubMed PMID 18657511 . Oligomerized...OPEN OPEN gGTCATCTGCcattcGAGGCGGACa REST corepressor 2 OZ553 and OZ554 OPEN OPEN gCTCACCATCtgttggaTGCGATGGAa...

  8. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine that sorts cells according to whether...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil...labeling T cells, B cells, and antigen presenting cells, scientists have been able to decypher cellular dynamics...tracked by cell sorter giving immunologists the ability to count cells and statistically analyze cell movements...fast T cells could jump from one antigen presenting cell to another. Indeed, we now know that T cells are...2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et al...

  9. AAVED

    Type
    Collection
    ...Regulation Ben Deverman 2:00 PM Cre-DOG and Combinatorial Strategies Connie Cepko 2:45 PM Break 3:00 PM (...GCaMP6f-P2A-nls-dTomato Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox...

  10. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    ...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...primary human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return...primary human cell editing, the researchers tested ABE8 in CD34+ hematopoietic stem cells. ABE8 efficiently... In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s...to edit primary human T cells efficiently makes it a promising tool for T cell therapy development.  Reducing...be introduced into cells as a plasmid or as mRNA constructs. In primary human cells mRNA delivery of ABEs...

  11. CRISPR Guide

    Type
    Collection
    ...Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell Stem Cell . 17 (2), 221–232. PMID: 26165924...used to transduce your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either...of CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa... regulation of transcription in eukaryotes. Cell . 154 (2), 442–451. PMID: 23849981 Gilbert, L. A., Horlbeck...Transcriptional Programs with CRISPR RNA Scaffolds. Cell . 160 (1–2), 339–350. PMID: 25533786 Cas9 alternatives...single RNA-Guided endonuclease of a Class 2 CRISPR-CAS system. Cell , 163 (3), 759–771. PMID: 26422227 Zetsche...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...

  12. Fluorescent Protein Guide: FRET

    Type
    Collection
    ...referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the...standard consisting of ECFP and EYFP connected by 2 GGSGGS repeats pET28CLY3 Peptide linker standard consisting...Protein Resources: Empty Backbones Biosensors Subcellular localization Optogenetics Background Förster ... Constructs to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses...

  13. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    ...PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells, 2013. 31(3): p. 458...possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition of reprogramming... somatic cells that are otherwise difficult to obtain. These can then be used to perform cell therapy ...in vitro. Find stem cell plasmids at Addgene Many sources of adult somatic cells have been probed for...Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells Int, 2016. 2016: p. 1329459. PubMed...

  14. Optogenetics AAV Preps

    Type
    Collection
    ...Constitutive 1, 2, 5, 9 Deisseroth 26973 pAAV-hSyn-hChR2(H134R)-EYFP Syn ChR2/H134R EYFP Constitutive 1, 2, 5, 9....WPRE.SV40 CAG ChR2/H134R mCherry Constitutive 1, 2, 5, 9 Deisseroth 127090 pAAV-CAG-DIO-ChR2(H134R)-eYFP...Optogenetics AAV Optogenetic tools can be used to control cell activity in living tissue with light. Optogenetics...

  15. Stem Cell Models for Disease & Open Science: Interview with Darrell Kotton

    Type
    Blog Post
    ...develop therapies to treat disease in the lung. Chapter 2: Balancing research with medical practice as an MD...engineered cells in therapy. The lungs are difficult, but other tissues like the retina have cell-based therapies...Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using stem cells from patients to...sharing reagents, cell lines, data, and knowledge without exclusivity. Chapter 11: The Stem Cell Bank (CReM ...the lungs, and, among other projects, using stem cells to develop in vitro models for lung disease. Darrell...what drives him as a praticing physician and stem cell researcher. Darrell's ultimate goal - to develop...means to treat disease through tissue repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary...

  16. Zinc Finger Consortium: Nuclease Expression Vectors

    Type
    Collection
    ..., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for expression ...vectors designed to express nucleases in different cell types. Zinc finger arrays can be transferred in-...vectors designed to express nucleases in different cell types. Zinc finger arrays created using ZFC reagents...nuclease domains: pST1374 (for expression in mammalian cells or zebrafish) pDW1775 (for expression in plants)... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM335... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish (can be used as a substitute for pMLM336... Miller et al., Nat Biotech 2007 ) in mammalian cells or zebrafish). pMLM802 (for expression of a ZFN ...

  17. Antibody Guide

    Type
    Collection
    ...surface of most immature B cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative... produced. Cells - Antibodies can also be produced in cell cultures using hybridomas, cell cultures created...created by fusing B cells with myeloma cells (immortalized B cells). The genetic reorganization that creates...

  18. CRISPR Plasmids - Epigenetics

    Type
    Collection
    ...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme... time and are potentially heritable in dividing cells. CRISPR Activate and Interfere technologies also...

  19. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    ...), 1668. https://doi.org/10.1038/s41467-019-09656-2 More resources on the Addgene blog CRISPR101: Validating...hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem cells can be implanted...Annotation of genes in immune cells typically involves the creation of germline knockout mice, which ...CRISPR-based systems enable gene knockout in immune cells in a high-throughput manner, but these systems have..., ubiquitous knockout of single genes in immune cells in vivo (LaFleur et al., 2019a and LaFleur et al...many genes have different functions in different cell types, at different times, and in combination with... L-CHIME Knocks out a single gene in defined cell types (lineage-based) pXPR_070 Violet-excited...

  20. CRISPR Plasmids - Tagging

    Type
    Collection
    ...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh...Protocols, 2016 . Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh...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...

  21. AAV Viral Preps

    Type
    Collection
    ...AAV vectors for trial purposes in serotypes AAV1, 2, 5, 8, 9 and retrograde Retrograde AAV To target projection... tools that effectively transduce many mammalian cell types. AAV is used in a wide range of experiments...Tetracycline Transactivators, Affinity Purification, Cell Ablation Monosynaptic Neuronal Tracing Rabies Virus-Based...

  22. Optogenetics Plasmid Collection

    Type
    Collection
    ...optogenetic tool is the widely used Channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii . ChR2 is a ... optical switch tools to measure and manipulate cells and their biomolecular processes. Plasmid...to blue light and allows cations to flow into the cell. Variants of channelrhodopsin have been engineered...improve the expression and membrane targeting in host cells. Other microbial opsins, such as the Halorhodopsins...actively move cations or anions into or out of the cell. When using these opsins as a tool to control neuronal...plasmids that use light to control a protein or cellular activity. For example, search "Cdc42", "transcription...

  23. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    ...(I), or sparse (S) labeling of the cell population. Figure 2: Dense (c), intermediate (d), and sparse...single genetically-defined subset of cells. By labeling fewer cells in a population, it’s easier to visualize...non-overlapping cells. While transgenic animals are commonly used to specifically manipulate a cell type of interest...expressed. In cells where the stop cassette remains, the effector is not expressed and the cells are not labeled...sparse labeling of the cell population that expresses SPARC and the fraction of cells that will be labeled... interest in an organism, all cells of that type are affected. Previous tools to overcome this restraint... of the gene construct. A predictable number of cells will express the effector or retain the stop cassette...

  24. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    ...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins...pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the host cellular receptor. To more...point for SARS-CoV-2 researchers to investigate how the virus interacts with human cells. Addgene’s mammalian...genes are important for SARS-CoV-2 entry into human lung epithelial cells (Biering et al., 2021). In their...Wesemann DR (2021) Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike bioRxiv. https://...labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman...

  25. Cre-lox system

    Type
    Collection
    ...Cre-ERT2 CAG Mammalian Heller 112622 pVHC_PGKneoLox2DTA.2 Venus, Cre-ERT2, targeting vector with MCS for homology...homology arms Mammalian Heller 112623 pEHC_PGKneoLox2DTA.2 Emerald, Cre-ERT2, targeting vector with MCS for homology... arms Mammalian Heller 112624 pmTHC_PGKneoLox2DTA.2 TFP, Cre-ERT2, targeting vector with MCS for homology...arms Mammalian Heller 112625 ptdTHC_PGKneoLox2DTA.2 tdTomato, Cre-ERT2, targeting vector with MCS for ...25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed . Ventura A, Meissner A, Jaenisch R, Jacks...allowing for direct comparison of Cre+ and Cre- cells. While Cre-lox recombination is a very useful tool...promoter like CAG, or expressed only in a subset of cells under a more specific promoter (e.g. Rho-Cre is ...

  26. Reagent Repositories Are Speeding up Science During the Pandemic

    Type
    Blog Post
    ...Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV-2 were developed for ...different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in this study came from...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...that science is truly a global endeavor. Case study 2: Plasmids Another example of reagents that have broad...applications but have proven useful to the study of SARS-CoV-2 are the lentiviral production and packaging vectors...

  27. CRISPR Plasmids - gRNAs

    Type
    Collection
    ...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...is highly recommended that you determine if your cell type contains the target sequence before using any...

  28. CRISPR Plasmids - Cascade-Cas3

    Type
    Collection
    ...are largely underutilized compared to their Class 2 counterparts like Cas9. This is attributed in part...bound to DNA, and the break can be repaired by the cell’s endogenous repair mechanisms. Cas3 belongs to the...

  29. Bikard Lab - CRISPR Repression Collection

    Type
    Collection
    ...chromosomal integration of DNA. ACS Synthetic Biology, 2(9), 537–541. DOI: 10.1021/sb400021j Figure 1: Multiplexing... CRISPR plasmids. Each point represents a single cell, and each color represents the population obtained...

  30. Resolute Plasmid Collection

    Type
    Collection
    ... funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777372....quality of research output, RESOLUTE expects to accelerate the pace of research in the field of SLCs to ...

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

    Type
    Blog Post
    ....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...that cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...in tables 1 and 2, with a cell-type specific promoter or enhancer now allows for cell-type-restricted ... overall (GFP+ cells that also stained for a neuronal marker). Check out tables 1 and 2 below for a summary...high dose to maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e...require the generation of a transgenic mouse. 2. Cell-type-specific restricted transgene expression in...

  32. New and Upcoming Viral Vectors - May 2020

    Type
    Blog Post
    ...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read... results, but also confirm expression in target cells. While the various AAV serotypes can be used to ...

  33. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    ...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...provide a 2-10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...

  34. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number of cell types at different...Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein (Plasmid #141391...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...

  35. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ...Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020. (B) Mapping...designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface. The Bloom lab used ...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...

  36. Gendered Innovations: Why Does Sex of the Cell Matter?

    Type
    Blog Post
    ...state-of-the-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how...never considered the sex of their cells. For one thing, cells and cell lines degenerate over time—and sex... effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to ...therapeutic capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—which...sexes in basic research is excellent design), but they used all female stem cells—this was an unconscious... stem cells. Nor did they detect important differences in function between male and female cells. The ...experiment. In the case of stem cells, these factors may include cell type, the disease being treated,...

  37. CRISPR References and Information

    Type
    Collection
    ...Link opens in a new window) An encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast and lentiGuide-Puro...interest and correspond to edited and non-edited cell populations. For more information, see the Hsiau...138 KB Musunuru CRISPRs in human pluripotent stem cells pCas9_GFP ; gRNA empty vector Protocol at StemBook...Bauer Protocol for Genomic Deletions in Mammalian Cell Lines pSpCas9(BB) (pX330) Protocol at Addgene Parrott...

  38. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ...interest in the cells you are studying. Read our SPARC blog post for more details. Figure 2: Dense (c... rising intracellular calcium levels within the cell. When levels of Ca2+ are high in the cell, calcineurin...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The... of a genetically-defined subset of cells. By labeling fewer cells in a population, it’s easier to visualize...lengths, the density of cells that are labeled can be altered to dense (45% of cells labeled), intermediate...or aging cell populations. Figure 3: Micrographs of wild type (top panels) and cells with high...barcodes (iBARs) in their loop region. After infecting cells with sgRNAiBAR library containing lentiviral vectors...

  39. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

    Type
    Blog Post
    ...Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 184:2372-2383...months, we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts...They were able to tweak this assay to study SARS-CoV-2 spike variants. “We created a series of spike expressing...relative ability to neutralize.”   Figure 2: Sera used in the study came from individuals who ...This sera was used in the high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was...viruses are quite genetically distinct from SARS-CoV-2 and its variants. Yet, the B.1.351 variant, which ...effectiveness of current vaccines against SARS-CoV-2 variants, this study was the first to compare so many...

  40. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate research and to help ...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

  41. AAV for Neuronal Tracing

    Type
    Collection
    ...G-protein is delivered to the starter cell. (2) The starter cell now expresses TVA and G-protein. EnvA-pseudotyped...spread from this cell to synaptically connected cells (meaning, it can spread to cells that are projecting...infect a single "starter" cell, but new virus cannot spread to any neighboring cells. To enable monosynaptic...provided to the starter cell in trans , via delivery by an AAV. Since this starter cell now has all the viral...projecting to the starter cell). Once in the secondarily infected cells that lack the G protein, the virus ...connected cells. To further refine the targeting of G-deleted rabies to specific starter cells, the tropism...is delivered to the starter cell, and specifically targets the starter cell via the EnvA receptor. RABV...

  42. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    ...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...understanding how SARS-CoV-2 interacts with human proteins. For example, we used HEK293T cells in our first study...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

  43. CRISPR Plasmids - Base Edit

    Type
    Collection
    ...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme... inosine, which is treated like guanosine by the cell, creating an A to G (or T to C) change. Adenine ...

  44. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ...this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen... hiPS cell lines generated using these plasmids, please visit the Cell Catalog on the Allen Cell Explorer... tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can... in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving..., and the stem cell lines we’ve made using them are available as part of the Allen Cell Collection at ...

  45. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...change the media (add 2-3 mL of fresh media once a week), and do NOT harvest the cells before at least 10...titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 cells and produce more rAdV ...infected cells show this “cytopathic effect.” Amplification can be repeated multiple times (2-4 rounds...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result in a 10...

  46. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1...spike protein’s role in infecting cell lines without using intact SARS-CoV-2 virus. Scientists have been using...Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi....production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid... pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details...

  47. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

  48. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...

  49. CRISPR Plasmids - Drosophila

    Type
    Collection
    ...plasmid Bullock and Port 49409 pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair ...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...

  50. 10 Basic tips for mammalian cell culture

    Type
    Blog Post
    ...: 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ...Make sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay...contaminants. Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine...’s best to split cells before the plates are more than 80-85% confluent because cells like their space...lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split very...numbers! When starting off with a new cell line, grow the cells, aliquot them into a number of vials, ...
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