...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A....joined Cell Press to pursue a career in science communication and publishing after completing her postdoctoral...

...fluorogen activating protein - dL5** in this case), and 2) a fluorogen (the TAP - targeted and activated photosensitizer...cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...PMID: 26808669. PubMed Central PMCID: PMC4916159. 2. Jacobson, K., Rajfur, Z., Vitriol, E., & Hahn, K....generation when bound by the dL5** FAP.  The iodine-modifications also shift MG-2I’s excitation wavelength to ...FAP-TAP system works. Find the Plasmids from the Publication Here Light-induced cell ablation with MG-2I-dL5...

...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...beforehand on which chromosome your docking site is located). This amounts to a small difference of two or ...throughout the lifespan of the fly to determine the implications of your genetic manipulations. You can produce...

...fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’re working with purified...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 ...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...new green fluorescent protein, some affinity purification tags, another version with that same red fluorescent...

...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ... it passes through and scatters in different directions. The degree of scatter is captured by detectors...Light that scatters sideways to the path that it was initially traveling is called side scatter (SSC) and...

... that universities require application submission in-house approximately 2 weeks before the RFA due date...requested in the call. Step 2: Develop a research plan Most grant applications restrict the space available...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and offers...the grant application process. When my PhD advisor first asked me to write a grant application due the ...in a previously funded grant application to see how funds were allocated. Step 3: Get feedback from your...the next round of applications. Step 5: Keep writing! Each written grant application builds upon the ... project grant application" by the NIH Still want more information on the application process? What types...

...2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...user-controlled illumination with violet light and (2) exposure to increased intracellular calcium. It has... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...the sensors discussed below fall into this GECI category.   For calcium sensors, one size doesn...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for ...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 ...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...

...PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal ... (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...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ...nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these...on-target and off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...

...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...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful...which can then be used for downstream cloning applications. While these two strains ultimately perform ...

...Quotes taken directly from participants (summarized in 2) touched upon many problems in this area: “ [The] ...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. McDowell, Gary S., et al. "Shaping the Future of ...A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed PMID: 24137543. Pubmed Central...University of Massachusetts Medical School. Graduate education programs are also beginning to address these issues...The panel discussion on Friday morning will be dedicated to Academic data and the Labor Market. On this...Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic Medicine...disease.  Outside the lab, he is interested in communicating science and in the intersection between science...

...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...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...steric and charge-based nature of the phosphoryl-modification; they are both much smaller, and carry only ...thus cannot reflect the true state of protein modification within the context of its appropriate regulation...

... 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, ...constructs. His work was acknowledged in hundreds of publications including Cell, Nature, and other journals. ...

...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...sequences via oligo synthesis using the format indicated below and pool-clone them into the pAWp28 storage...realization of a variety of perturbations and applications in functional genomics, cell reprogramming, ...CRISPR/Optogentics Tools Resources on Addgene.org Catch up on Your CRISPR Background with Our CRISPR Guide...

...labeling of delicate axonal and dendritic processes not previously visible with Brainbow-1 and -2. The general...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...Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition to improving...temporal control without the need for germline modification, and enables Brainbow to be used in a variety...technique will provide important insights into the complicated physical organization of the brain and other ...

...of other Cas proteins found in the so-called class 2 CRISPR-Cas systems. There are 24 variants of the cas14...the presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific...of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this viral sequence in the assay...

... Step 1. Steps 2 and 3 are usually very easy — our tech transfer team will communicate with your organization...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first...species can be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid...name and match the name as it appears in its publication or as it is commonly known in your lab. Plasmid...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside...reagent/antibody, unspecified, or other. If other, indicate type in “Vector Type Other” column. For example...

...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the wild-type promoters...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression...

...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable...Change in Undergraduate Biology Education: A Call to Action, Washington, DC. 2. President's Council of Advisors... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... research into undergraduate courses and in communicating science to the public. You can find out more...

...Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators for Ca2+ based on green ... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...sufficient spectral overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately...decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance energy transfer by monitoring...have enhanced the ability of labs to answer sophisticated questions about cellular processes. Currently...