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...splitting my cells first. Time management through planning I probably wouldn’t have fallen into my cell splitting...for Scientists. Back when I used to do mammalian cell culture, I would often respond to friends asking...there in a few minutes, I just need to split some cells.” Inevitably, this task would take longer than I...since my days as an undergrad working with cancer cell lines. As I started planning out my own experiments.... In my anecdote above, I prioritized splitting cells over meeting up with friends, but didn’t realize...mean in terms of the utility of your tasks. In my cell splitting anecdote, I probably could have grabbed...with friends and come back to the lab to split my cells later. Eating with friends has a somewhat immediate...

...Neuroscience at Harvard University. Just as computers, cell phones, and cars become more technologically advanced...blotting, restriction mapping, and sequencing gels, as well as their modern equivalents and see what we can ...gene mutation is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with... of the mutated β globin gene that causes sickle cell anemia. The mutation happens to destroy a sequence...conducted on a sample from an individual without sickle cell anemia, two DNA fragments are observed because the... site where MstII cuts. In a patient with sickle cell anemia the enzyme does not cut the DNA so only one...strand (8). Adding a small amount of a single radiolabelled ddNTP to a synthesis reaction along with all...

...This novel tool is practical in cell and organ culture, as well as cell lineage studies. Eisenhoffer and...CreLite for cell tracing using the transgenic zebrafish model, ubi:zebrabow. In this system, cells express...such as the brain, eye, muscle and epithelial cells, as well as in different stages of development, ensuring...fast-splicing split intein and co-infected into the cell. Intein splitting removes any exogenous sequences...split into two plasmids and co-transfected into cells for reconstitution. Image from Levy et al., 2020...red-light inducible optogenetic tool for precision in cell lineage tracing Article contributed by Erin Sanders...fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene that recombine...

...Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System.” Cell 155 (2013): 1379-1491. Pubmed...of 6 chromosome-specific loci in the U20S cancer cell line./ Each color represents a gRNA targeting a ...localization and movement of this locus in live cells! This technique was developed in the lab of Addgene...et al. to track telomere dynamics throughout the cell cycle. To optimize the signal to noise ratio of ... Green PP7-PP7 Red boxB-boxB Cyan MS2-PP7 Yellow PP7-boxB Magenta boxB-MS2 White boxB-MS2-PP7...different hairpins will produce a secondary color cyan, yellow, or magenta, depending on the hairpin combination...gRNAs with two different hairpin sequences produce yellow, cyan, or magenta light. A gRNA with all 3 distinct...

...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, ...CRISPR-mediated control of gene repression and activation." Cell 159.3 (2014): 647-661. PubMed PMID: 25307932. PubMed...Genes and Genotype-Specific Cancer Liabilities." Cell 163.6 (2015): 1515-1526. PubMed PMID: 26627737. ...Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library." Nature...

...Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Osuka, S., Isomura... includes the HNH and RuvC catalytic domains, as well as the CTD (also known as the PI), while REC includes...colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix, purple. ...   Fun Fact! Even though the Cas9:RNA complex is well described in the closed conformation, recent electron...structural disorder. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix, purple; sgRNA...efficiency and are important for robust activity in cells (Jiang & Doudna, 2017). Within an interior cavity...from that structure. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge helix, purple; sgRNA...

... into a mouse embryonic stem cell (ESC) line, enrich and select for cells that had successfully incorporated... when it gets exciting – the cell sends out an “SOS” signal and cellular repair mechanisms rush in to ...Cas-Mediated Genome Engineering. Cell 154:1370–1379 . https://doi.org/10.1016/j.cell.2013.08.022 Yoon Y, Wang ...Cas-Mediated Genome Engineering. Cell 153:910–918 . https://doi.org/10.1016/j.cell.2013.04.025 Zhang X, Liang...develop into chimeric mice (with a fraction of the cells in the adult mice derived from the engineered ESCs...file. Then I saw it, the chromatogram showing the telltale signs of a mutation introduced by CRISPR. The ...immune system in bacteria and archaea. As you know well if you’ve been following the Addgene blog, it has...

...its oxidized derivatives. Cell 152, 5 P1146-1159. https://doi.org/10.1016/j.cell.2013.02.004.  Xu X, Tao...targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc.2016.9.   ...interacting with transcription machinery.   Well, I’ve got some good news: complete DNA demethylation...enzymatic activity cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy...methylate sites blocked by dCas9. Within a few rounds of cell division, the original methylated strand is diluted...restrictions, anything within 5 base pairs should work well, and up to 10 base pairs away (especially 3’ of ...dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can...

... be introduced into host cells, such as bacterial, yeast, or mammalian cells, for antibody production ...Nanobodies and recombinant binders in cell biology. Journal of Cell Biology 209:633–644 . https://doi.org...animals and bleed dates and even clonal hybridoma cell lines can produce more than one mAb (Bradbury et...plasmid or from an integrated sequence in a stable cell line. The genes encode the heavy chain and light...antibody protein binds an antigen. Image from Fvasconcellos.     How are Recombinant Antibodies Made...to create antibodies to targets that may not work well in animals because of similarities to host proteins...light chain recombinant monoclonal antibodies as well as other forms of antibody-based (e.g., nanobodies...

...CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed...characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern...editing than Cas9, confirming that this protein is well suited for genome editing.  Find Cpf1 Plasmids at...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-... smaller than SpCas9. These nucleases should fit well into AAV vectors, especially given that Cpf1 gRNAs... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing...

... systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...Integrated in Parallel. Cell, 154(4), 914–927. https://doi.org/10.1016/J.CELL.2013.07.018 Anzalone, A....epigenetic manipulation of prime editing. Cell. https://doi.org/10.1016/j.cell.2024.03.020 Mathis, N., Allam, A...suited for prediction of very diverse edit types and cell contexts. Figure 1: High-throughput screening...train ePRIDICT (Figure 3), a model that predicts how well a genomic site can be edited based on its chromatin...https://doi.org/10.1126/sciadv.aax9249 Koeppel, J., Weller, J., Peets, E. M., Pallaseni, A., Kuzmin, I., Raudvere...double-strand break repair pathway balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/...

...the algae cell is just the cell wall and cytoplasmic membrane. Vampirovibrio chlorellavorus will not grown...bacteria Vampirovibrio chlorellavorus is a predatory bacteria that attaches to the cell wall of green algae...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al.... (Soo et al., 2015). There, it divides into two cells. At the same time, it sucks out its contents (hence...Grasshopper parasite The parasitic worm Spinochordodes tellinii‘s larvae makes its home inside grasshoppers and... the predatory cyanobacterium Vampirovibrio chlorellavorus." PeerJ 3 (2015): e968. PubMed PMID: 26038723...

... the green, red, yellow, cyan, blue, and orange spectra. A comprehensive list of cell markers all tagged...of fluorescent proteins and their use for imaging cell biology, Michael Davidson’s lab at Florida State...matching a relatively easy proposition, explained Michelle Baird, who has created plasmids as a technician...From F-Actin to mitochondria, from the nucleus to cell membrane, this collection has fluorescently tagged...of many proteins known to localize to specific cellular structures.The Davidson lab even included some... where your protein of interest localizes in the cell, use these markers as your guide. As Michael Davidson...Fluorescent proteins are] a whole new way of seeing cell biology, seeing what interacts with what, what affects...

...expansion depends on how well the gel components penetrate the sample, how well the gel polymerizes, and... for EM is not something you find in the average cell and molecular biology lab, and EM is limited in ...ultrastructural relationships that influence everything from cell division to memory, but don’t have access to the...fix (and maybe label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample...properties of the sample itself. For example, cultured cells are typically easier to expand than mouse brain ...techniques, and adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al...., Mohar B, Eddison M, et al (2022) Visualizing cellular and tissue ultrastructure using Ten-fold Robust...

...reach essentially every cell or else small pools of antibiotic resistant cells could repopulate the environment...strategies are nonspecific - they harm any bacterial cell without a resistance gene, allowing resistant bacteria...antibiotics target one of three essential processes: cell wall synthesis, DNA replication, or protein synthesis...eliminated from the bacterium, which may also induce cell death. CRISPR also enables increased specificity...Treating a mixed population, represented here by cells of different colors, specifically targets bacteria...bacteria. Targeting a genomic sequence results in cell death. Plasmid targeting may result in plasmid loss...and antibiotic resensitization, or may also cause cell death. Image from Beisel et al., licensed under ...

...to amplify fluorescent signals. Named for the "stellar explosion SUperNova," SunTag can help you turn ...time to get enough signal. This workaround risks cellular phototoxicity and eliminates the possibility of...potential to form aggregates and may be toxic to the cell. Here comes the SunTag How does SunTag fix these...enables single molecule tracking  within living cells without affecting protein function, thereby creating...creating a single-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed some...attempted single-molecule imaging deep inside the cell, in the nucleus and cytoplasm. Again, they found...SunTag constructs would avoid negative effects on cell physiology. Having seen that mitochondrial tracker...

... light sensitive proteins (opsins) to manipulate cell activity, enables researchers to silence or incite...organism because their larval stage lends itself well to studies of neuroscience. The larvae of zebrafish...channels that allow charged particles to cross the cell membrane when exposed to light. Neuronal firing ...channel LiGluR both cause ion movement that results in cell depolarization. They therefore function as a neuron...in negative chloride ions which hyperpolarize the cell. It therefore functions as a neuron “off switch....stimulus. While the escape response neural pathway is well characterized, there is much left to be discovered...They found that photostimulating the Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement...

... out of 10,000 cells on average! Without some means of quickly determining which cells successfully received...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...witin 1 month for optimal efficiency. Beware of satellite colonies! Carbenicillin is more stable than Ampicillin...

...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this...sequence. That means there are 61 possible tRNAs. Cells however, may not express all 61 of these tRNAs and...may be found at very different levels (Mauro and Chappell, 2014). Due to this variation in tRNA expression... are also optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized the C...sites and even affect protein function (Mauro and Chappell, 2014). Different rates of translation by different...and in biotechnology and therapeutics (Mauro and Chappell 2014).  Ultimately, it is important to take a ...//doi.org/10.1186/s12859-017-1793-7 Dresios J, Chappell SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-...

... SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible...with such techniques can be problematic, however. Well designed aptamers can help reduce such background... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting temperature...cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further enhance...