...fluorescence-activated cell sorting (FACS). This is not possible in plants outside of limited, single cell protoplast...genes known to be essential for cell growth. Together, this excellent work highlights the importance of...visually identified subpopulation. For traditional cell culture, enriching your active RNAi subpopulation...where he investigated how plants achieve polarized cell growth in the lab of Luis Vidali. For his next adventure...Screens for Effective Gene Silencing in Plants. Plant Cell 25:1507–1522 . https://doi.org/10.1105/tpc.113.112235...Augmin in Phragmoplast Microtubule Generation. Plant Cell 24:1478–1493 . https://doi.org/10.1105/tpc.112.098509...dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of...

...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...the PAM. With this information, they turned to a cell culture system to see if any Cpf1 nucleases would...would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. The application of Cpf1 to genome...RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. Find plasmids from...

...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...spreading in non-target cells, including yourself as the researcher.   Figure 1: Separation of retroviral...re derived from viruses that can infect people, cells, and animals. Some of these viruses are completely...While the virus can still enter (and thus infect) cells, this safety feature prevents the virus from replicating... the procedure. Mice move much more quickly than cell cultures do, and tired researchers are accident-...these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that...lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus...

...4,000 cell lines. Seventeen of these cell lines were used to investigate the susceptibility of cells from...additional cell line used in this study came from the Japanese Collection of Research Bioresources Cell Bank...that SARS-CoV, which entered human cells via hACE2, could not enter cells expressing murine ACE2 (Subbarao.... Figure 1: Biological repositories that distribute mouse strains, plasmids, cell lines, and much...from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate ...accelerating research into this virus. One such example is cell lines. The American Type Culture Collection (ATCC...of this pathogen. The sharing and distribution of cell lines from repositories on opposite sides of the...

...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different...separately.  Mutant cells are enriched using either drug selection or fluorescence-based cell sorting and screened...mutant cells can be used in drug screens to identify genes that confer drug-resistance. Mutant cells are ...generate lentivirus (B). Cas9-expressing cells or wild-type cells are treated with lentivirus containing...plus Cas9 to generate the mutant cells, respectively (C). Mutant cells are screened (D) and "hits" are ...lead to skewed results. Selecting a cell type - Theoretically, any cell type can be used in a CRISPR screen...requires a massive amount of cells as starting material. Therefore, cell types that are of low abundance...

...estimated to affect anywhere from 1 in 6,000 to 1 in 19,000 live births (1).  While disruption of the smn1... funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...plasmids to upregulate their expression in specific cells. With a more concrete understanding of the molecular...molecules targeting SMN1 and its partners in mouse cell lines. Dr. Di Schiavi hopes that these studies will... of ~30 patient derived peritoneal mesothelioma cells lines and assess their tumorigenesis. These experiments... cause spinal muscular atrophy, the molecular mechanisms behind the disease are unclear. Dr. Di Schiavi...

.... Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Sander JD, Cade L, Khayter...coming closer to this DNA targeting ideal. Figure 1: (A) Representation of a zinc finger (ZF) nuclease... domain, a ZF array is an effective targeting mechanism for molecular tools. ZFs are not without their...

...endocrine cells. It follows that the specific targeting of these voltage indicators needs to be cell type ...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet...Indicators As previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is ... specific cell types is where viral vectors become invaluable. Viral vectors are mechanisms by which ...electrical events! Voltage indicators are voltmeters for cells! In this blog we will review how these indicators...related to the electrical impulse of the cardiac cells. To study this phenomenon you will likely want to...the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling is carried out by...

...fluorescence images of C2C12 cells stably expressing pTRE-Tight-MitoTimer and rtTA. The cells were pulsed with 2µg... Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1;126(Pt ...neighboring cells in imaging applications and the mapping of neuronal projections to their associated cell bodies...stem cells concomitant with activated DsRed2 expression. Brown et al., Genesis 2014 Jan;52(1):39-48... to this system by engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing... targeted to subcellular regions or protein complexes for proteomic mapping in live cells. Lam et al...been used by several groups to monitor protein and cell ageing, as the spectrum shift occurs during Timer...

...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...Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell Tropism after Delivery to... 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...

... can rupture E. coli spheroplasts (cells which have had their cell walls removed with lysozyme). This ...function, we might be able to endow cells with new ways to interact mechanically with their environment.   Jessica...protein fusions allowed us to image R bodies inside cells, and these constructs may be useful to future scientists...applications. For example, you could use them to lyse cells (and surrounding membranes) in keeping with their...bioengineering, but it wasn’t clear whether they could 1) be decorated with proteins and remain functional,...Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. ...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...

...injecting a cell suspension to create xenograft models. Restraint: back scruffing or mechanical restraint...different substances can be injected, including cells, drugs, DNA, viral vectors, and viruses. All researchers...scruffing or mechanical restrainer Back or hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site... advanced and finally, to expert.   Figure 1. Visual representation of common injection routes ...back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner Intradermal ...work. Good luck with your in vivo experiments! Table 1: Summary of common injection routes in mice. Injection...resources on the Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms...

...Virus (MoMLV, MMLV, MuLV, or MLV) or Murine Stem Cell Virus (MSCV) genomes whereas lentiviral vectors ...things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic diseases...vectors, their infectivity is limited to dividing cells. Find tips on using gamma-retroviral vectors in ...are able to transduce dividing and non-dividing cells. More detailed descriptions of the lentiviral elements... pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an RNA-mediated gene silencing mechanism. Scientists... to express siRNAs of their choice in mammalian cells.  Learn more about Mammalian shRNA Tools for RNAi...-optimized Cas9 under the control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown...

...organs, cells comprise compartments and structures, called organelles. Take a sneak peak inside a cell with...proteins, however, in this case the cell needs to be fixed and the cell membrane permeabilized in order for...Cooper GM. 2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory...with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role in...involved in intracellular transport pathways, it is possible to map the internal organization of a cell. This...for labeling your subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection...biochemical methods thus removing the context of the cell. However, much knowledge about functionality can...

...polymerase gene or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes...choose cell lines that are highly transfectable.  The amounts of each plasmid transfected into cells are ...capable of being replicated and transcribed in the cell when the appropriate viral proteins are provided... RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...on separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome. These...the minigenome) are efficiently transfected into cells. All of these components are necessary to efficiently... RNA polymerase also needs to be present in the cells to transcribe the viral mRNAs and minigenome. This...

... for Mapping Multiprotein Complexes in Human Cells.” Cell Rep. 13: 621-633. PMID: 26456817. Find plasmids...Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half...locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus to verify...crispr.mit.edu. They tested their system in HEK293 cells, integrating the GFP-Puro cassette into the FBL ...homologies. Abbreviated protocol for PITCh Step 1: Generate microhomologies in the PITCh vector ~20...editing possibilities MMEJ can enable. References 1. Sakuma, Tatsushi, et al. (2016).  “MMEJ-assisted ...CRISPR-Cas9 with the PITCh systems.” Nat Protoc. 11(1): 118-33. PMID: 26678082. Find the plasmids from ...

...CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed...RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227.   Potential ...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. What we still don't know about Cpf1...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016 Jun 6. PubMed PMID: 27272384...specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology (2016). PubMed PMID: 27347757...

...several excellent reviews have collated data from various papers to make the decision easier. 1. Müller... monitoring sensitized emission in living plant cells." (2013). PubMed PMID: 24194740. PubMed Central ...-molecule FRET for imaging biomolecules in live cells (Sustarsic and Kapanidis 2015), which may lead to...understanding biomolecular structure and dynamics in live cells." Current opinion in structural biology 34 (2015...acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation by absorption of a photon... overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately for...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...

...– just 1 out of 10,000 cells on average! Without some means of quickly determining which cells successfully...detail selection in non-bacterial cells such as yeast or mammalian cells.   Name Class Mode of Action...Inhibits cell wall synthesis Bactericidal 100-200 ug/mL Carbenicillin beta-lactam Inhibits cell wall ...the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to its own...easily detect plasmid-containing bacteria when the cells are grown on selective media, and provides those...kill bacteria (bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction...down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite ...

... is technically cell-permeable, its use in live cells can sometimes result in cellular toxicity due to... Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental context...Additionally, cell permeability is a critical factor, as it influences the dye's ability to enter cells without...enabled the visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive...interfere with protein expression or function in live cells. Fluorophore properties: The specific properties...context of the experiment, such as the presence of cells or specific proteins, can influence the choice of...ligands is unaffected by additional protein from cells under certain conditions, which is an important ...