...approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells (Milo, 2013)...about RapID! EXCELL: Proximity labeling for the detection of cell-cell interactions EXCELL is an enzyme-mediated...enzyme-mediated proximity cell labeling strategy to detect cell-cell interactions in vivo (Ge et al., 2019...et al. "Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell–Cell Interactions." Journal...a bait protein fused to BirA* in cells and then incubate those cells with exogenous biotin. After a few...peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed on the surface of a cell of interest, where...-protein interactions, and even more recently cell-cell interactions. If you’re looking to identify interactions...

...positive cells by the number of only mCherry positive cells (Martin et al., 2019). Figure 2: Editing...restoring mCherry expression. Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the...easily determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry...expressed in cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T ...TREE) to purify single base edited cells without the need for single cell isolation and downstream sequencing...fluorescence and cell sorting using FACS which could limit their use across different cell types and systems...sgRNA targeting the GFPACG to the cells. As expected, only the cells that contained the cytidine base ...

...of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...RNA aptamers are used to track cellular processes by transforming cells with plasmids encoding aptamer-tagged... been used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ...and later introducing the small molecule into the cell. The fluorescence that is generated allows for qualitative...). DFHBI is membrane permeable and non toxic to cells. Because it rapidly photobleaches, DFHBI can only...

...PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating human somatic cell gene knockouts...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata from...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...endogenous allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed ...

...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression... want to make sure your cells have first reached an appropriate density. Cells in the exponential growth...collecting dead cells, and, conversely, you cannot induce too early as there are not enough cells in the culture...host protein, using up valuable resources in the cell thus leading to slowed growth. Additionally, some...such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

...interesting things to do, feel free to crowdsource! 2. Free food generally gets people to show up If you...picniced outside of our new office space on the nicely manicured lawn of University Park at MIT.  I’m ...

...community is active, engaged, and has great questions! 2. The r/Science subreddit is well moderated. One of...questions from the AMA - as you’ll see below, it pretty nicely displays her energetic, truthful and helpful style... training is abundant, helpful, and sometimes excellent. Finally, as I said in the AMA, it’s incredibly...topics to the Addgene blog The blog has many other excellent posts on science career topics as well. Instead...

...start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid, while... highlighted below the table.     Figure 2: This decision tree can help guide your choice on ...transfer to the adult brain. Nature Biotechnology, 34(2), 204–209. https://doi.org/10.1038/nbt.3440 Goertsen... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...mouse to primate neocortex. Cell Reports, 34(13). https://doi.org/10.1016/j.celrep.2021.108754 Ravindra Kumar...

... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...in HIV-1 infected T-cells to see if they could be rescued by CRISPR. CD4+ T-cells were isolated from healthy...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

...variety of environmental conditions, and double every 2 hours. Yeast are also the first eukaryotic genome ... properties and processes with our own cells. Like human cells, yeast DNA is packaged into chromosomes...differentiation of β-cells in the the developing zebrafish pancreas. They first tagged these cells using plasmids...recapitulate the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than...than studies in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study...to identify a set of mutations that made cancer cells more resistant to chemotherapy. You can find these...to optimally control gene expression in specific cells of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces...

... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...

...Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d Ref BphP-based...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...the concentration of BV-containing molecules in a cell and depends on the efficiency of BV incorporation...

..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...proteins enable the observation of proteins in living cells and organisms. Both components of the chimera are...However, these may not reflect the situation in cells. Therefore, the localization of critical fusions...currently the standard for assessing oligomerization in cells. In addition to measuring stickiness, several researchers...fluorescent proteins that behave as monomers in mammalian cells include: mTurquoise2, mEGFP, mNeonGreen, mScarlet...protein to the existing pool of protein within a cell and may lead to over-expression artifacts. This ...heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32. PubMed PMID: 21619590...

...Nutr, 129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic...carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier...chemically. Enzymatic biotinylation occurs naturally in cells and is the process by which biotin is covalently... Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides to a substrate...substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for...proteins in bacteria, yeast, insect and mammalian cells. Check out Kay et al., 2009 for methods on biotinylating...Application of avidin-biotin technology to improve cell adhesion on nanofibrous matrices.” Journal of Nanobiotechnology...

...of concept for further development. Figure 2: Mechanisms of deaminase-based adenine transversion...base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j.molcel.2024.01.021 Tong, H.,... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. The...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...

...two visible blue or magenta chromoproteins (Figure 2) (Liljeruhm et al., 2018). This system is a nice confirmation... that converts sucrose into a toxic metabolite. Cells that have undergone a double crossover will not ...contain sacB, allowing them to grow on sucrose. Cells that have only gone through the single cross over...Giacalone et al., 2006). The Chen lab demonstrated excellent counter-selection which enabled very efficient...desired deletion directly into cut vector with excellent efficiency using a commercial isothermal assembly... from Harvard. Jake received his MD and a PhD in Cell and Molecular Biology from the University of Pennsylvania...

...as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should include...to detect dead or dying cells so that you can base your analysis on living cells only (for example, propidium...This tends to be most problematic when staining cells or tissues that are the same species as your primary...propidium iodide only stains dead cells). Compensation controls address the spectral overlap that occurs...Quantitative Fluorescence Microscopy.” Journal of Cell Biology, vol. 185, no. 7, Rockefeller University...

...Postdoctoral Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective... can treat the underrepresented individuals too nicely. When the work of the underrepresented individual...

... Methods 2009; 6, 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey...is immediately ready to transform into competent cells. The master mix of enzymes can be purchased from...mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically ...

... from damage (Abe & Lim, 2024).    Figure 2: A) When proteins (blue) interact with the air-water...epigenome editing. Cell, 184(9), 2503–2519.e17. doi: https://doi.org/10.1016/j.cell.2021.03.025.    Recombinant...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs contain... will only be transiently present in transduced cells, limiting off-target editing. Cargo size limits ...01133-w Pandey, S., Gao, X. D., Krasnow, N. A., McElroy, A., Tao, Y. A., Duby, J. E., Steinbeck, B. J.,...site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime ...

...improvement in muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice...human T cells and hematopoietic stem and progenitor cells (HSPC)s to ~40% expression in the K562 cell line...gene to airway epithelial cells in mice. This vector transduced ~10% of cells, similar to the transduction...Using AAV Donor Vectors. Cell Reports 20:750–756 . https://doi.org/10.1016/j.celrep.2017.06.064  Carvalho... then independently packaged into an AAV. When a cell is co-infected with both part A and part B AAVs,... always.  The combination of serotype and tissue/cell type could have a big impact on the rate of transgene...since both of these variables influence whether cells will be co-transduced with multiple copies of both...

...signal separately for each target.   Figure 2: Examples of simultaneous multiplex IF labeling of...immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC) with tissues), where...know if they co-localize at the same place in the cell? Or you know that the presence of several biomarkers...tissue sections, methanol- or paraformaldehyde-fixed cell samples, or other preparations. Sequential IF For...hippocampus, or counting the number of puncta in each cell. When making figures for a paper or presentation...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...

...15.4%) 46 (59%) of these students were in the last 2 years of their degree. Students filled in a pre-program...professional and personal development, is essential to excelling in the modern world. Talent knows no boundaries...

...Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv. ...on or in these cells: antibodies can bind to proteins on the cell surface, or the cell membrane can be...antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens such as parasites...fusing B cells that have been extracted from animals injected with protein antigen to myeloma cells. The ... measurement of protein expression in cell lysates, whole cells, or tissue samples. Many experiments that...heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA can be used to quantitatively...same cell simultaneously as well as compare between samples. Learn more about flow cytometry. Cell and ...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced....experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms... efficiency. Finally, as shown in Drosophila S2 cells (Böttcher et al., 2014), inactivation of non-homologous... community. Developments in mammalian and yeast cells using modified Cas9 proteins to regulate gene expression...homologous recombination donors in cultured Drosophila cells. Nucleic Acids Research 42:e89–e89 . https://doi.org...

...experience in cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in...that, when those contracts fall through or are cancelled, then redundancies can be made within the company...

...ask new and interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the ... optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance...

...biology 14.6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure...ligase bound to nicked DNA-adenylate." Molecular cell26.2 (2007): 257-271. PubMed PMID: 17466627. 25. Luo...electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes...