...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...antibody-encoding genes over time as the hybridoma cells divide. These changes can alter the antibody that...

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

...experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms...loci. The first approach – co-CRISPR from Craig Mello’s lab – used inactivation of the unc-22 gene as their... efficiency. Finally, as shown in Drosophila S2 cells (Böttcher et al., 2014), inactivation of non-homologous... repair templates (Farboud et al., 2019).   The Mello lab also reported that modifying the 5’ modification...., 2020). Together, these improvements from the Mello lab resulted in strikingly high editing efficiencies...selectable markers.  CRISPR in C. elegans The Meyer and Mello labs also identified several experimental set ups...Cas9 editing (Farboud et al., 2019). Work from the Mello lab has dramatically improved RNP editing with linear...

... that are diffused or actively transported from cells, traverse the body through the bloodstream, and ...understanding of how these processes are regulated at the cellular, tissue and organ level by nuclear receptors (...corepressors depending upon a context defined by ligand, cell type, and promoter, organizing the expression of... coactivator proteins individually contribute to cell regulation in a coherent manner. Because coregulators...that coactivators may be integrators of multiple cell signaling systems. Activation of membrane receptors...sense the impact of the total environment on the cell. The NURSA Consortium Now in its third phase of ...and disease states, their circadian regulation, as well as in important physiological states such as adipogenesis...

...number of cells have been edited. For many experiments, the end goal is to generate a clonal cell line, so...doi.org/10.1093/nar/gky164 Cancellieri S, Canver MC, Bombieri N, Giugno R, Pinello L. CRISPRitz: rapid, high-throughput...designed your gRNA and introduced it into your target cells with Cas9. Hooray! Now it’s time to make sure your...blog post we’ll explain how to verify that your cells were appropriately edited for your desired mutation...important to know a few things about your target cells and the desired edits. First, you will need to know.... Validating knockout mutations When a knockout cell line is desired, any mutation that introduces a ...input is sequencing trace files of WT and edited cell lines. Then decomposition analysis is performed ...

...persist over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR... high percentage of muscle cells throughout the body, especially stem cells 2. Remove any immunogenicity...patient myoblasts; when injected into DMD mice, these cells expressed functional dystrophin. Editing genes in...in vivo is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and...be delivered to both cardiac and skeletal muscle cells, where precision editing of the dystrophin gene ... concept treatment, as the disease is especially well suited for genome editing. The homology-directed... the percentage of dystrophin-expressing muscle cells increases over time, and all three groups confirm...

...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...

...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,...controls the cell types in which the conditional recombination can occur based on the cells in which Cre... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...

...spread. They can be inherited by daughter cells during cellular reproduction, similar to the host’s chromosome...elements in the human gut. Cell, 187(5), 1206-1222.e16. https://doi.org/10.1016/j.cell.2024.01.039 Garcillán-Barcia...transfer can occur naturally through transformation (cellular uptake from the environment), transduction (viral...bacteriophages), or conjugation (direct transfer from one cell to another). Bacteria need special fertility genes... of a plasmid benefiting its host. For example, cells with a penicillin resistance plasmid can grow in...also carry the necessary resistance genes to help cells survive their own secretions. Pro tip! Historically...bacteria invade hosts, including attaching to host cells and evading the host immune system, and those that...

...currently a Postdoctoral Fellow in the lab of Mary Jeanne Kreek at the Rockefeller University.  His interests...considerations? I recently spent two months in the lab of Antonello Bonci, Director of the National Institutes of ... to implement the technique in my lab at The Rockefeller University. In this and upcoming posts in this... has already been proven. Therefore, a concrete, well defined, and robust behavioral output is sufficient...opsin expression in the intended brain region and/or cell type (I’ll talk more about this in detail later)...desired optogenetic construct in the brain region and cell type of interest is obviously of critical importance...through the vector cores of various universities as well as Addgene. See the Deisseroth lab homepage for ...

...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...

...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...

...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...

..., 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...

...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...

...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...

...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...

...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...

...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, ...

...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...

... 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...

... 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...

...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 ...

...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...

...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...

...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 ...

...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 ...

...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 ...

... 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...

... 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...