...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...

... 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... delivery vehicles with great safety profiles as well as other benefits. What makes AAV vectors great...

...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 ...

...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...

...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...

...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...

...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...Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in NYC. The CRISPR/Cas9 system is ...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...

...CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via FACS, primer validation...article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized...CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations...deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given locus ...screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones...methods for CRISPR design, cloning, and delivery, as well as their techniques for creating deletions, identifying... His lab studies the molecular genetics of blood cell development and disease. Learn more at his webpage...

...help in the capture of other rare cell types, like stem cells or cells infected with malaria or other parasites...capture cancerous cells expressing low levels of tumor antigen. I asked Howarth to tell us more about SpyLigase...looking at, magnetic isolation of circulating tumor cells, we found that the binding stability really mattered... in the pili that they use to anchor on to host cells. Addgene: Were there any major hurdles along the... donor for keeping going while we worked out the cell capture system.) Cancer and other applications Addgene...cancer? Howarth: The capture of circulating tumor cells (CTCs) is going to have a big impact on our understanding...Addgene. Howarth: We submit all our plasmids that may well be of use to others to Addgene. I am a big fan; ...

... competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared using...plasmid that transforms well in other competent cells) to ensure that your cells can be readily transformed...to make your own competent cells It’s easy to make your own competent cells. If you need a high efficiency...purchasing competent cells. However, for most everyday cloning, homemade competent cells will work just fine...Preparing chemically competent cells (protocol)  Preparing electrocompetent cells (protocol)  Untergasser...of chemically competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene...Set  from Zymo Research to make competent cells because cells prepared using this kit can be transformed...

...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...expressed. Each cell remains the same color for its entire lifetime and daughter cells retain the same... the fate mapping of cell populations over time. The ability to track single cell dynamics at the organism...expressed in a single cell maintains its color over the entire length of the cell, even axons of up to ... specific cell type. The Confetti mouse was first used to describe the self-renewal of cells in the intestinal...for tracking the fate of Langerhans cells. In the Ubow mouse, cells expressing YFP, CFP, and RFP are not... of CFP++ cells permitted researchers to longitudinally map the turnover of Langerhans cells in the dermis...

...purification as well as reversibly regulation of endogenous signaling activity in cells.  Let’s shed some...OptoNBs to control cell signaling The second application for OptoNBs takes us into cells: light-based control...their use for protein purification and controlling cell signaling. Key components of OptoNBs OptoNBs have...can bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes... over-activated in cancer due to its involved in cell proliferation and differentiation.  Since Ras is...They expressed this OptoNB-SOS fusion protein in cells that expressed membrane-localized mCherry. With ...With darkness, SOS binds Ras and initiates the extracellular signaling-regulated kinase (ERK) signaling cascade...

... life that remained unexplored. Using new single-cell sequencing approaches, the lab was able to eliminate... and so the lab organized a sampling trip to Yellowstone National Park (YNP). Extremophiles - phylogenetically...sample was separated into smaller samples of 5-10 cells each for sequencing, allowing for lower complexity...

...transfected cells, we count the number of tag-positive cells (tag ICC) as a percentage of total cells in the...pre-assembled in a 96-well plate. Glioma stem cells were transfected in 96-well format using an Amaxa ...Deliver csRNP into cells: Prepare cells for transfection, add csRNP and ssODN to the cell suspension, transfect...Proteins - the workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical...epitope tagging in mammalian stem cells. For easy scalability to the 96-well format, we developed a “TAG-IN...csRNP plus ssODN, is then transfected into stem cells. Cells are allowed to recover for 2-5 days and then...efficiently these components are delivered into cells; for each cell type, the transfection/delivery step needs...

...researcher at Imperial College London, Dr. Tom Ellis. Dr. Ellis does research in synthetic biology and bioengineering...and Addgene  Many thanks to our guest, Tom Ellis! Dr. Ellis is a lead researcher at the Centre for Synthetic...yeast chromosome. Listen to learn more about Dr. Ellis, synthetic biology, synthetic chromosomes, and obstacles...academic researcher. Listen to the interview with Tom Ellis Podcast breakdown Intro: How Tom ended up in academia...techniques. Chapter 4: Research projects underway in the Ellis lab including yeast and E. coli engineering, microbial...specialising in engineering gene regulation in yeast. Dr. Ellis is now one of the world's leading yeast researchers...Resources at Addgene.org Find Plasmids from the Ellis Lab Find Synthetic Biology Kits Browse Our Synthetic...

...where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells could also...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...degradation within labelled cells limits the timespan available for tracking certain cell types. Another ...or off in cells expressing Cre. The result of a system like this is a mosaic of cells labelled with different...

...Induction of Human Germ Cell Fate from Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94....differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(...blood cells, and renal epithelial cells from urine are some of the easily isolated somatic cells that ...some of these cell types [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal...differentiated cell to a different cell type. While transdifferentiation is suited for switching cell types between... differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010. 7(5): p. 618-30. PubMed...et al., Reprogramming of T cells from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. PubMed...

...primary human cells  Cell therapy approaches can use base editors to correct mutations in stem cells and return... a way to improve editing in primary human cells (Gaudelli et al., 2020). One of the widely used base ...base editors with improved editing in cell lines Nicole Gaudelli, the Associate Director and Head of Gene...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...base changes in cellular DNA and RNA dependent or independent of the gRNA used (Gaudelli et al., 2017)....

... the single-cell level can aid in drawing connections between signaling activity and cell phenotype. One...Covert, M.W. (2018). Live-cell measurements of kinase activity in single cells using translocation reporters... One method for monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging...their Kinase Translocation Reporters (KTRs) whose cellular localization serves as a proxy measurement of ... used to measure kinase activity in specific subcellular compartments. Second, because KTRs shuttle between...when designing KTR experiments. Third, KTRs are not well suited for monitoring kinase activity that occurs...

...vectors (right) are transduced to target cells. Within target cells, Cas9 machinery is transiently expressed...-transformed mammalian cells. These viral vector systems also work equally well in dividing and quiescent...in order to facilitate its entry into the target cells (Beatty et al., 2012). These features make AdV an... complexes into transformed and non-transformed cells yielded rates of targeted mutagenesis similar to...nuclease-induced gene disruption frequencies in the various cell types ranged from 18% to 65% (Maggio et al., 2014... leads to a vast majority of AdV-modified human cells being subjected to scarless homology-directed genome...quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral vectors Since then...