...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively... not well understood, researchers have been using AAV vectors for gene editing in mammalian cells since...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...since 1998. In that year, Russell and Hirata from the University of Washington reported a surprising finding...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...composed of single stranded DNA, which is particularly well suited for strand invasion, a key step in the homologous...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...

...packaging cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not...using helper-free packaging cell lines This method utilizes a packaging cell line that stably expresses... packaging cell line. For example, Phoenix, a second generation γ-retrovirus packaging cell line developed...and rat cells), or an amphotropic envelope, Phoenix-AMPHO (for the infection of mammalian cells.) Using...transduce both dividing and non-dividing cells because once in the host cell cytoplasm, the viral components are...for efficient gene delivery to mammalian cells using 293T cell-based systems. Curr Protoc Immunol. 2001...γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted the development of multiple...

... of cell types, so they’re ideal for work with primary cells or other difficult-to-transfect cells. Lentiviral...dividing cells. Another type of vector, based on adenovirus, could infect non-dividing cells, but without...turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first lentiviral vector system...ability to transduce both dividing and non-dividing cells. Lentiviral vectors are the vector of choice for...viral envelope to dictate the tropism, or types of cells the vector could infect. Finally, the transfer plasmid.... After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral particles...adeno-associated viral vectors can also target non-dividing cells, lentiviral vectors can carry much more genetic ...

...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted to ...reporter output was observed in their 293 cell culture system. In cells cotransfected with GFP and the GBPs,...expression was induced in ~76% of GFP+ cells, and 100% of DsRed+ cells also expressed GFP. Using AAV constructs...cortex, cerebellum, and spinal cord. Cre-DOG is also suitable for optogenetic studies, and since cells infected...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed ...wanted to express a variety of proteins in single cell types in the mouse, but knew it would take years...could then selectively manipulate only GFP-labeled cells. Once Tang and Cepko found a description of GFP-...

...currently available from Addgene. Rosella autophagy biosensor Rosella is a dual-emission autophagy biosensor... autophagy, Rosella is targeted to the vacuole in yeast or the lysosome in mammalian cells. This decrease... and cytoplasmic Rosella. When fused with a mitochondria-targeting sequence, Rosella can be used to track...al. "Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast...process by which cytoplasmic material, including organelles, are targeted to lysosomes for degradation. Autophagy...provides limited information about what materials or organelles are being targeted for autophagy. The development...). In the absence of autophagy, the untargeted Rosella remains in the neutral pH cytoplasm where it emits...

...pulses to the subject/cells expressing the optogenetic tool causing a change in cellular activity or gene ...receptors (GPCR) regulate cellular function through multiple intra-cellular signaling cascades. Optical... in the extracellular domain which activates the native signaling cascade of the intracellular part of...positively charged ions to rapidly pass through the cell membrane and initiate an action potential - a very...have developed many optogenetic tools to control cellular activity with exquisite precision using only light...both the membrane potential and a host of other cellular functions. Photoswitches are engineered molecules...such as Na+, K+, and Ca++ to passively enter the cell with the concentration gradient to depolarize it...

...and inside the cells and are thus typically used for distinguishing live from dead cells. The combination...the chart tells us that PE and PE-Cy7 are "very bright" when they are excited by the yellow/green laser...say I am culturing HEK 293T cells in vitro. I transduced the cultured cells with a virus that encodes a...panel. Besides some background cell death constantly happening in cell culture, the transduction process... to some cell death. As a result, there is a high abundance of positively stained dead cells present and...When analyzing your cells using flow cytometry, you are typically measuring the presence or absence of...certain markers on the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence...

...fluorescence-activated cell sorting (FACS) helps researchers sift through thousands of cells to identify cells exhibiting... CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ from Biggiephage can ...target single cells. Now, Francois St-Pierre’s lab developed a new form of FACS-based cell screening, Single-cell...individual cells with unique spatiotemporal phenotypes from large heterogeneous cultures.  HeLa cells expressed...was used to image over 3 million cell types expressing variants of Yellow Fluorescent Protein (YFP). The...Cre for use in bacteria CRISPR-Casɸ mGold, a new yellow fluorescent protein New CRISPR plasmids New items... conformational changes in a protein.    Cells expressing an RFP reporter plasmid for recombinase...

...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (2013). ...patient’s own cells that have been modified ex vivo, previously edited healthy donor cells can be delivered... editing these cells to avoid a negative immune response (e.g., graft vs. host) as well as the introduction...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (2013). Gerald Schwank et al. ...on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis ...delivery directly to the patient. Patient-derived cells can alternatively be harvested, edited ex vivo, ...

...on freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically...organism. Furthermore, cell free systems can be freeze-dried into pellets (FD-CF pellets), leaving them shelf-stable... into a host cell (through a process called transformation), essentially giving the cell all the instructions...to dispose of the live cells. The alternative is to express the protein in a cell-free manner. This is ... about operating the rest of the car as well. Similarly, cell free expression systems contain all the ...first molecular biology lab in college that I really fell in love with it. Something about being able to actually...biology, especially those at the molecular and cellular level. The reason for this is cost and complexity...

...targeted One Many Sample type(s) Cell lines Cell lines, primary cells Table 1: Key differences between... of cells (~5x107). Together, these two requirements prevent the use of CAPTURE with primary cells or ...need to create stable cell lines and allows CAPTURE 2.0 to be used with primary cells. Find plasmids for...that occur during development and cellular differentiation. Using a cell line model, the Xu Lab tracked ...by Biotinylated dCas9. Cell 170:1028–1043.e19 . https://doi.org/10.1016/j.cell.2017.08.003 Liu, X., Chen...CAPTURE method requires the creation of a stable cell line that co-expresses three components: dCas9 ...drawback of the original CAPTURE method is that a new cell line has to be created for each genomic loci of ...

... specific cell-type promoter. Once injected into the brain, the viruses infect all the cells with an inactive...the brain remained elusive, not from a lack of intellectual curiosity on the part of scientists but, rather...energy conversion or the induction of intra- or intercellular signaling pathways (Ernst et al., 2014). Microbial...proteins control the flow of electric charges across cell membranes and maintain membrane potential in response...once a certain number of positive ions crosses the cell membrane, a threshold is reached and the neuron ...selective expression of opsin genes in targeted cells makes it possible to control neuronal activity with...that, in turn, trigger neural excitation, whereas yellow light activates halorhodopsins to silence neuronal...

...Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with cell-based method ...Requires large cell population, senstive to time since cell fixing HTGTS Not reported Cell-based Identifies...method IDLV 1% Cell-based Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods... use single cell clones for in vitro CRISPR experiments. The likelihood that a single cell clone derived...and cannot be applied to heterogenous populations (Bell et al. 2014). Further, Sanger sequencing has a lower...product sequence. Targeted amplicon sequencing doesn’t tell the whole story Figure 1: Schematic representations...picture of the impact of CRISPR editing in the model cell line or organism. Several options exist for unbiased...

...introduced the reporter into the cells, you’re ready to edit. These cells are then transfected with several... up, all successfully transfected cells will express RFP while cells containing the reporter gene that...cut and repaired by NHEJ as well. The ratio of RFP to GFP expressing cells provides a real-time read out...using the RFP:GFP ratio, edited cells can be sorted from unedited cells. In an experiment that targeted...Activity. Frontiers in Cell and Developmental Biology 6: . https://doi.org/10.3389/fcell.2018.00054 Sander...CRISPR experiment was successful in your organism or cell type? You can use DNA sequencing or other molecular...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...

...dividing cells only, so is not suitable for infection of quiescent cells or infrequently cycling cells but ...non-dividing cells, making it conducive to targeting all cell types. Altogether, AAV has many excellent features...DNA copies of their genome upon infecting a host cell. This viral DNA (also called proviral DNA) is then...host. So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily optimized gene...and permanently integrate a genetic cargo into a cell. These viruses have a cargo capacity of ~ 8 kb, ...needs, and can transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus...but is fine for experiments with immortalized cell line cultures. Lentivirus Lentivirus is a type of retrovirus...

...looking at an intracellular or extracellular molecule? By fusing a biosensor to an organelle-specific targeting... fluorescence across a population of cells (i.e. in a plate well). Use to track a process (e.g. enzyme...Localization within cells Fluorescence microscopy Visualize components of individual cells Separation/identification...fluorescently-labeled components Fluorescence-activated cell sorting (for cells); fluorescent HPLC (proteins or other molecules...) Isolate or quantify cells, proteins or other molecules that have been labelled with your fluorescent...signal. Blue flame GECO biosensors from Robert Campbell’s lab display calcium-dependent increases in fluorescence...by Olivier Pertz’s lab. Other biosensors track cellular characteristics like pH, redox state, and voltage...

...animation.)  Many types of immune cells, from mast cells to B cells, express Fc receptors that bind to...) antibody-dependent cellular cytotoxicity (ADCC) c) antibody-dependent cellular phagocytosis (ADCP). ...opened in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive when holes...die.  Antibody dependent cellular cytotoxicity (ADCC) Antibody dependent cellular cytotoxicity (ADCC) is...(NK) cells expressing Fcγ receptors binds to IgG and release cytotoxins that kill the target cell. If ...response, by binding to Fc receptors on other immune cells. (psst! Need a quick review of the different immune...pathogen and physically prevent it from entering a cell, a non-Fc-dependent effector function known as neutralization...

...resistant cancer cells exist prior to drug treatment. They infected a non-small cell lung cancer cell line containing...same parent cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate... target cell population at low multiplicity of infection (MOI) such that each individual cell receives...researchers to individually label millions of mammalian cells through lentiviral infection and to monitor their...quantify the take rate of mouse xenografts after cell implantation. Their goal was to figure out how to...ClonTracer library to determine whether or not cancer cell populations resistant to drug treatments arise during...They hypothesized that, if they labeled the parent cell population using the library, propagated it, separated...

...structure or protein is located within a cell. The Goedhart lab, Gadella lab, and collaborators have an ongoing...for labeling specific structures within cells. HeLa cells were transfected with plasmids encoding...affinity), as well as GRAB_NEmut as a negative control. Find the sensors at Addgene! New yellow fluorescence...protein evolution Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system...selection in the cytosol (instead of on the yeast extracellular surface) which enables tracking of protease ...mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image from Mateo...labeling structures and compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent...

...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells with the ...of a cell being infected by only one viral particle, but it also increases the chances that a cell doesn...adequately represented in the cell population, you’ll need to infect many more cells than the number of plasmids...appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus. Purification of...small aliquot is not enough to transfect producer cells, so your first step will be to amplify the library...individual plasmids knows, some plasmids amplify well and others don’t. The size and composition of your...

...improved cell-free system to examine biosynthetic pathways A new tool for enriching ABE-targeted cells The ...Improved cell-free transcription/translation system to examine biosynthetic pathways  By Gabrielle Clouse...interest, and cell extract.    Graphical Abstract: Schematic representation of cell-free system,...successfully reconstituted in this cell-free system. In summary, this cell free system allows for a relatively...   New tool for enrichment of ABE-targeted cells By Ellie Adams Single base modifications through ABE...multiplexed and can be used in human pluripotent stem cells, a cell type that has been difficult to modify in the...Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems) have been...

...produced in E1-containing mammalian cell lines such as HEK293 cells. 1st generation AdV cloning capacity....*Pro-Tip*: The cells will become completely confluent and the media will turn yellow. Do NOT change the...more HEK293 cells and produce more rAdV to reach higher titers. Successfully infected cells will become...large transgenes, transduce quiescent and dividing cells, and do not integrate into the host’s genome. The...administration, which results in the death of transduced cells and loss of transgene expression (Interestingly,...gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine...can be generated if the E1 gene from the packaging cell line is transferred into the AdV by recombination...

...including Trends in Cell Biology, Cell, Molecular Cell, Developmental Cell, and Cell Stem Cell.  She was the...inception and launch of other Cell Press journals, including Cell Metabolism, Cell Reports and Joule. In September...Deborah Sweet from Cell Press. Deborah Sweet is the Vice President of Editorial at Cell Press.  She trained...the founding Editor-in-Chief of Cell Stem Cell, a role she held for 10 years, and played a major role in...by Deborah Sweet, Vice President of Editorial at Cell Press. Almost everyone who works in a lab struggles...and making sure you do what you can to help your fellow researchers advance science overall. Communication...communication of science very seriously. That’s why at Cell Press we developed STAR Methods to improve the structure...

... brain, robust single-cell labeling is necessary to distinguish individual cells. The Brainbow-1.0 construct... May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... both of Harvard’s Department of Molecular and Cellular Biology, harnessed these attributes of Cre-lox... 3.2. Brainbow labeling has also been applied to cell marking and lineage tracing in other systems, such...Brainbow highlights the trend in biology towards single-cell resolution imaging. Sanes and Lichtman endeavored...contains three fluorescent proteins: RFP (red), YFP (yellow) and M-CFP (membrane tethered cyan). Without Cre...caused partially by fluorophore photoinstability, as well as the tendency of fluorophores to aggregate in ...

...PFs need to tell and sell their stories to get jobs. PFs (and mentors) can never be too well practiced ...doing a postdoctoral fellowship, what are you signing up for and how can you do it well? Below, I briefly...exciting story I can tell with my data?  Can I make better slides that more effectively tell a story? Generally...Hughes Medical Institute. Today's postdoctoral fellows (PFs) face a number of challenges ranging from ...briefly describe some tips for a successful postdoc fellowship. 1. Reverse engineer success On Day 1 of your...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary...need to be there? A typical biomedical postdoc fellowship lasts five years. Most universities and institutes...

...specific cell type rely on transgenic models that express recombinases like Cre or Flp in their cells of interest...entry into the cell (Dudek, AM. 2018).  If the serotype you are using transduces cells that you are not... an ATG start codon to prevent transcription in cells where the recombinase was not present (Kuhlman, ...recombination that can lead to leaky expression in cells that do not express Cre or Flp. Here at Addgene ...recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally, we have experienced.../Flex vectors and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about...volume needed to achieve adequate expression in your cells of interest.  The AAV serotype used can exhibit ...

... cultured cells as well as more difficult conditions such as model systems and primary cells. References...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... human CRISPR libraries. Brunello: human CRISPRko sgRNA library The Brunello library consists of 77,441...criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled library...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion... viability screens. Based on these metrics the Brunello library was more effective than other popular ...genes. In the subsampling analysis of the sgRNAs, Brunello even with only a single sgRNA outperformed another...

...measure, and control molecular signals, cells, and groups of cells in order to understand their activity...Fluorescence-activated cell sorting (FACS): This is a type of flow cytometry that separates mixtures of cells into distinct...Thus, FACS can be used to separate cells expressing GFP from cells that are not. Developmental/transgenic...(GFP) has likely existed in creatures such as jellyfish for millions of years; however, it took until ... localization profiles, acting as a biosensor or cell marker, studying protein-protein interactions, visualizing...consisting of 238 amino acids derived from the crystal jellyfish Aequorea victoria. It has a fluorescent emission... was non-toxic and could be expressed in living cells, enabling the study of dynamic, physiological processes...

...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...pol, and env), the isoforms of these proteins, as well as the viral long terminal repeats (LTRs) are different...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...

...successfully infected/expressing cells by flow cytometry. The isolated cells were injected into mice and imaged...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...acceptor. NLuc is an excellent BRET donor due to its bright luminescence. Intracellular Calcium Sensor Yang...to changes in intracellular calcium and is useful for direct imaging of events in cells and brain explants...could “clone by phone”. One of my professors was excellent at phone cloning with labs around the world and...luciferase by Promega’s Advanced Technologies group as well as evolution of the coelenterzine substrate to the...