...researchers facilitate and accelerate scientific discovery. How can Addgene help accelerate your research? Let...Addgene’s executive director, said during our celebratory lunch, it’s hard to picture one million plasmids...assuming 400 boxes fit in a freezer). Our Addgene celebration was so much fun! Here's the Addgene Leadership...

...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, ... Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

... a wide variety of cell lines, from HEK to CHO cells to neural and endocrine cells such as AtT-20, PC12...then handled. Additionally, many cell lines, especially the endocrine cell lines we most commonly work with...specific cell line, and its transfection efficiency. We often trypsinize the transfected cells on the day...increased). For some cells, add a drop of serum to aid in outgrowth. Depending on the cell type, we sometimes...growing cells such as HEK and CHO do not seem to require this, while nearly all endocrine cells do. While...difficulty deriving single clones from SH-SY5Y cells, as these cells did not seem to form discrete colonies but... of Medicine. In the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently...

...of Multiple Kinase Activities in Live Single Cells." Cell 157, no. 7 (2014): 1724-734. PubMed PMID: 24949979...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to ... dimeric Dronpa is less prone to aggregation in cells compared to the tetrameric Dronpa it was derived...inhibitors, psMEK1 and ERK KTR-mRuby2 expressing cells were incubated with an inhibitor of choice, exposed...inactivation, and then decreases. Treatment of the cells with a PP1/PP2A inhibitor leads to maintenance of...Raf-MEK-ERK signaling in the hypodermis and gut cells of C. elegans causes the same phenotype. Activation...signaling pathway with psRaf1 or psMEK1 in these same cells by 24-48 hours exposure to blue light also induced...

...that already do mammalian cell culture. HEK-293T cells are the most common cells used in viral production...versus insect cells for viral production Consideration Mammalian cells Insect cells Viruses AAV ...type of cells that will be used to make the virus. Viruses can be produced in many different cell types,...produced in bacteria cells. However, some viruses can be produced in multiple different cell types, including...you make an informed decision. Mammalian cells Mammalian cells are a great option for viral production,...production in mammalian cells is relatively quick. After transfection, HEK-293T cells start producing virus...using qPCR or digital droplet PCR. Insect cells Insect cells are a less common option for the generation...

...varying across cell types. Variance can be caused by cell cycle rates; rapidly diving cells will have higher... of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle, when...HDR events, ensure cells are actively cycling through the cell cycle stages. If cells are overly confluent...way to make a knock-in cell line is to utilize the built-in repair pathway cells already have for repairing...than quiescent cells. It is important to know that HR is often mutated in cancer cells, specifically in...keeping an eye on those cells! To take this one step further, you can inhibit cell cycle regulatory proteins...negative outcomes on viability if cells are arrested too long. Prolonged cell cycle arrest can lead to apoptosis...

...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... Podcast Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients... 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...sharing of his OSKM factors have affected the stem cell community – a community largely created due to Yamanaka...

...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 ... and images generated from these cell lines can be found in the Cell Catalog on our data portal. The plasmids...

...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...benchmarked on positive control genes in CD4+ and CD8+ T cells. To demonstrate the power of these systems to investigate...

... do so, their T-cells were grown in vitro, their mutations corrected, and the T-cells were transferred...functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside the...a wide range of cell types including but not limited to muscle, liver, and brain cells. There are a multitude...chain (c) and is characterized by a lack of immune cell development and function. To date, gene therapy ...that can deliver gene therapies directly to the cells that need them. Adeno-associated virus (AAV) – based...delivery of nucleic acid cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery...expression over many years in postmitotic, long-lived cell types. AAVs in the clinic Thus far, AAV vectors...

...HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing cells treated...inactivate target proteins or cells while having little effect on neighboring cells. The photosensitizer used...Here Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG...effective killing of dL5** cells without death of neighboring bystander wild-type cells.   Zebrafish experiments...fewer mCer3-labeled cells in their hearts 24 hours post treatment and no mCer3 cells present 96 hours post...increase cell ablation efficiency, but dL5** phototoxocity is induced when expressed on the cell surface...protein inactivation/cell ablation while having limited collateral damage to bystander cells. Are you ready ...

...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...tool that allows for the labeling of a subset of cells with the same genetic makeup, overcomes all of these...

...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with...vesicles via the secretory pathway to their subcellular or extracellular destination. At the end of their life...involved in intracellular transport pathways, it is possible to map the internal organization of a cell. This...proteins, however, in this case the cell needs to be fixed and the cell membrane permeabilized in order for...for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection...and selected subcellular marker proteins, it’s possible to get an idea about the subcellular structures ...Medicine, 7). Identifying the subcellular localization and composition of cellular complexes are important ...

...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,...sex of cells and tissues—when that information is reliable—is important to ensuring excellence in basic...

...easier, cost effective and faster; this has helped accelerate scientific discoveries.” Occasionally, a plasmid...dedication to scientific sharing, we wanted to celebrate our depositing PI’s who have the most highly requested...

...2020 coming up on March 7th, what better way to celebrate open data sharing than with Addgene’s AAV Data... expression after AAV injection, what region and cell class ended up expressing your virus? If you were... good of growing this open data resource and accelerating reproducible research. Share your data on AAV...

...sequence change the cell must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway...incorporated into the edited genome. In mammalian cells such as stem cells, HDR is relatively rare and DSBs are predominantly...them into somatic cells affected by the disease and study what’s wrong with the cells. However, our excitement...sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9 com­plex is very efficient... stranded breaks (DSBs) into genomic DNA in many cell types and often results in biallelic modifications... After genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we...excitement turned into frustration when we sequenced our cells and found how inefficiently our edits were being...

... plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we hope researchers ...poorly classified function and a well established cellular signaling gene, thus providing a starting point...reference cDNA clones mapping to well-annotated cellular pathways). It wasn’t clear whether gene-agnostic...and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome this first challenge..., we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely available to ...sequence-confirmed series of clones corresponding to known cellular signaling pathways as part of our Genetic Pathway...Mutations. Berger AH, Brooks AN, Wu X, et al. Cancer Cell, 2016. PubMed PMID: 27478040. The lung cancer mutant...

...gain access to some cells and tissues, such as human induced pluripotent stem cells (hiPSCs). Nanoblades... into particles at the cell membrane. Mature Nanoblades then bud into the cell culture supernatant and... Addgene. Genome editing primary cells with Nanoblades Primary cells are often refractory to gene delivery...mouse bone marrow (BM) cells, primary human hepatocytes, and hematopoietic stem cells (HSCs) using Nanoblades...-70% for these four cell types (Table 1). Additionally, hiPSCs and mouse BM cells still behaved like iPSCs...Primary cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM cells from GFP...mouse BM cells when using a gRNA targeting the Fto gene, while editing was not detected in cells electroporated...