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...protein localization, as well as cellular processes like gene expression and cell migration (Sladitschek... MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with strong...High-throughput cloning, in a nutshell, is the systematic combination of different genetic sequences ...imaging or flow cytometry applications in mammalian cells. The modules used in this method include fluorescent...components, one can generate constructs to visualize cell cycle dynamics, titrate inducible transgene expression...detection of each fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave...

... for stem cells, or metastasized tumor cells, or alzheimers-affected neurons, or [your cell line of interest...critical to stem cell survival. Knowing which genes influence the survival of our cell culture is the foundation...minimal side-effects on healthy cells and maximal effect on unhealthy cells. Until recently, the human genome...ENCODE project, find enhancers in embryonic stem cells, and conclude with a targeted CRISPR screen that...essential transcription factor in embryonic stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant...We start by infecting millions of embryonic stem cells (ESCs) with a lentiviral vector, an attenuated retrovirus...The result is a heterogenous population of stem cells, usually housed in a single flask. Through a bit...

...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...CRISPR in adult stem cells has huge potential, because the genomes of adult stem cells in organoid cultures...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank...connected to the early onset of Alzheimer’s disease, as well as those that play into late onset AD (5).     ...homologous recombination in cultured intestinal stem cells of patients with cystic fibrosis (8).   The difference...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis....is a big advantage over induced pluripotent stem cells, which need to go through a phase of crisis where...

...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...an inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...follow the same initial mechanism as in mammalian cells — by excising the freshly edited U bases — but E...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. Beyond...

...thought to activate programmed cell death or a dormant state for bacterial cells that have been infected with...collateral RNA cleavage response is well described in bacterial cells, LwaCas13a does not display nonspecific...eukaryotic cells. They arrived at this finding through analyses of global RNA expression, cell stress, and...they identified 53 potential candidate genes that fell into 3 categories based on the architecture of the...target RNAs, although its usefulness in bacterial cells will likely be limited by the propensity of Cas13a...pool of RNAs) or studying RNA processing in live cells. Indeed, groups have utilized dCas13 fusion proteins...genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID-19 (Joung, Ladha...

... immunology, cancer-immune cell interactions, cancer immunotherapy, as well as new approaches to reprogramming... fields of Cancer, Inflammation and Immunity, as well as various combinations thereof (so called “Immuno-Oncology...cancers. It was inspiring to participate in the 2017 Cell Symposium on Cancer, Inflammation and Immunity and...immunosurveillance. The presented study analysed a model of hepatocellular carcinoma, a cancer which can develop from ...example the upregulation of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment...follow the interactions between tumor and immune cells in real time. Addgene depositor Professor Max Krummel...effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system - COX inhibitors like aspirin...

...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...

...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 ...

...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...

...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...

...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...

...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...