...expression tTA Off Verma 26803 pEnt L1L3 EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving...systems were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested...Hermann Bujard tested the tet system in a mammalian cell system (HeLa) and found that the tet system was ... interest rtTA or TetR expression plasmid Stable cell lines can be made that continuously express a system...derivatives can be viewed in Krueger et al, 2004. Some cell culture sera may contain tetracycline or its derivatives...of gene expression at a single copy in mammalian cells; Expresses firefly luciferase hairpin and GFP under...vector lentiviral Gateway RNAi system for conditional cell line generation; contains expression cassette for...

... will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement...spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...two different ways 1) your hypothesis is wrong or 2) there is some problem with the technical implementation...PMID: 21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...optogenetics, ICSS can now be done with neuronal cell-type and temporal precision. In ICSS, a test chamber... remote contextual memories. This study is an excellent example in which the authors started with a brain... research. Derek P. Simon received his PhD in Cellular and Molecular Biology from the University of Michigan...

...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine deaminase...available for plant systems. Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful...based CBEs are known to deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer....PMID: 27096365. PubMed Central PMCID: PMC4873371.  2. Nishida, Keiji, et al. "Targeted nucleotide editing...homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery...Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an original C-G...’ to ‘I’ (inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic...

...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...Drosophila cell line, I’ve studied a transcription factor important for T cell differentiation (a cell type ...regeneration and homeostasis in the Drosophila midgut. Cell. 137, 1343–55 (2009). PubMed PMID: 19563763. PubMed...et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. ...

... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ... the EMX1 locus in human embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target...off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity... researchers to make precise edits in mammalian cells and may decrease worries about off target effects...

...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...populations (Figure 2). An immunologist who studies B cells, might then gate the B cell population and create..., these cell types will separate into distinct populations on a FSC versus SSC plot (Figure 2).  A user... Figure 2: Due to differences in their size and complexity, cells from a whole blood sample will...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...plot of CD3 (T cell marker) versus CD19 (B cell marker) to separate the B cell and T cell into distinct...separate the B cell population into progenitor B cells, immature B cells, plasma cells or others.   ...

... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...product, the transformation may not result in any cells, or all the colonies screened might not contain ...verify the strain and the efficacy of your competent cells (when they get old, they lose competency). If all... look at your experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is ...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide... Transform TOPO Cloning Reaction into Competent Cells: You can use your standard lab protocol for this...

...for Multiprotein Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...Bewersdorf, J. (2016). Two-colour live-cell nanoscale imaging of intracellular targets. Nature Communications,...opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure represents...

... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...service to further contribute to our mission to “accelerate research and discovery.” Here are the details...

...ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...

...illumination with violet light and (2) exposure to increased intracellular calcium. It has been applied to...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version named...

...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...genes essential for West-Nile-virus-induced cell death." Cell reports 12.4 (2015): 673-683. PubMed PMID:... (and multiple) genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems ... build and constantly improve gRNA libraries, excellent resources with effective gRNAs are publicly available...perturbations and applications in functional genomics, cell reprogramming, and beyond. For further details, ...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials... protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of...disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant...which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB...broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon...Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly...

...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained... 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...

...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...is observed in the absence of genome editing (Fig. 2 and 3) (5, 3). Benefits of truncated gRNAs Importantly...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome... genetic loci within a single cell. One might imagine transfecting cells with both the nuclease-positive...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as ...reliance on orthogonal Cas9 species. Within a single cell, full-length guides targeting one set of genetic...knockin mice for genome editing and cancer modeling." Cell 159.2 (2014): 440-455. PubMed PMID: 25263330. PubMed...

...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ...better than a nitrocellulose membrane. If you have already run your blot using nitrocellulose, you may decide...protein of interest in a complicated solution like cell lysate. But they can be a lot of work — and what...instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution...

... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the... most relevant to plasmids: Genetics & Heredity, Cell Biology, Biotechnology & Applied Microbiology, Biochemistry...

...phosphorylation." Nature cell biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., ...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...commonly cited that approximately one-third of cellular proteins are modified through phosphorylation ...translation of many stop codons and detrimental effects on cell growth. The C321.ΔA strain was genomically recoded...is particularly interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion...

... living plant cells." (2013). PubMed PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...fluorescent proteins available. Fortunately, several excellent reviews have collated data from various papers... alters the FRET efficiency. Detecting certain cellular changes with an intermolecular probe is often ...Miyawaki et al., 1997), was designed to measure intracellular calcium and published in 1997. Since that time...of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments can probe...