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...used. The Fujii lab demonstrated this in mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag...studies (Fujita et al., 2018). In addition to the "in cell" form of this method, in which an engineered DNA-binding...molecule such as the CRISPR complex is expressed in cells to be analyzed, the Fujii lab also developed another...Protein A, 1xFLAG, 2xAM, and HA have been reported, as well as affinity purification with an anti-Cas9 antibody... disease for which there is currently no cure (Campbell et al., 2018). Find plasmids from the Fujii lab...see our interview with Dr. Fujii. References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing...recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed PMID: 26848818. Fujita...

... into a pEcCas/pEcgRNA system. This system works well for engineering single genome edits but the double...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new pEcCas/pEcgRNA system We ... to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image... E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites edited. We optimized...crRNA. The spacers of the crRNA can be customized as well, including concatenations into crRNA arrays targeting...

... the same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent...conjugated antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody...the primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody ...antibody can be used. In a competitive ELISA a multiwell plate is coated with a competing antigen (green...enzyme labeled antibody and then added to the multiwell plate. The more antigen in the sample, the less...method, users immobilize the target antigen on the multiwell dish and incubate it with a reporter conjugated...ELISA, the sandwich ELISA, users first coat a multiwell plate with a capture antibody and then add their...

...into the cells. But for C. roseus, Agrobacterium infiltration into leaves does not work well, as the leaves...tumefaciens strain GV3101(pMP90), which works well for us. Use a well working vector system This allows you to...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...interest (Unver and Budak, 2009). This method works well in C. roseus. A common method for overexpressing...transient transformation systems for other species as well. Things to consider in developing a transient leaf...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...

...relied on for transducing cells: galactose and the AAV receptor (AAVR). Cell lines lacking galactose were...Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6 (2007): 1339-1347... Wilson labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B...commonly used to enrich adult mouse hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A...out of Ly6a in primary C57BL/6J brain endothelial cells decreased transduction rates while overexpression...overexpression of the C57BL/6J version of Ly6a in 293T cells enhanced PHP.B transduction rates. The group also determined...PHP.B, virus still reached the brain endothelial cells of these mice, although at lower levels than in ...

...Xenopus frog cell lines to develop robust methods for frog gene editing. They edit in a cell line, then ...is the smallest photosynthetic cell in existence, but most abundant cell on earth (twice the human biomass...Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to study for convergent evolution because...Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs are great for studying parenting... currently for terrestrial frogs. Cnidarian-Dinoflagellate symbionts (Virginia Weis) Sea anemone Aiptasia...killing frogs all over the world. These are less well known fungi (they are not yeast or mushrooms) but...Hawaiian bobtail squid has an extreme number of nerve cells and an unusually high level of RNA recoding in the...

...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...available to bind DnaA. Fun fact! As far as we can tell, DnaA doesn’t need to hydrolyze ATP to do its job...

...with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt...shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases,...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed...exciting to see future studies enabled by this tool as well as additional potential improvements to the editor... editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY...western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein ...

...dynamic samples Ex: monolayers of live cells with fluorescently labelled, motile structures These samples are...high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (<15 μm) tissue...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter whether... 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, small model embryos >15 μm Thsee are some...shouldn’t be afraid to seek out expertise from a well-established microscopist at your institution when...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional ...similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy. Nature methods 2,...

...published, followed soon by GCE done in mammalian cells (Shandell et al., 2021).   How it works If you’re interested...with) the cells’ own natural translation machinery; these are often incorporated into the cells via plasmid...so complicated? Well, while the techniques are straightforward, the systems – the cells growing and producing...is usually added to the media for uptake by the cells. Second, you’ll need tRNAs and tRNA synthetases ...biosynthetic pathway for the ncAA integrated into the cell’s genome, instead of incorporation via plasmid or...Theoretically, if you put all these together, the cell will produce a protein with a site specifically ...component of the experiment system (including media, cell type, and growth temperature) may need to be individually...

...have become indispensable tools for molecular and cell biologists over the last two decades, but their ...light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be...quantum yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent...produce photons. The original nano-lantern emitted yellow-green light and exhibited 3-5 fold increased brightness...three luminescent proteins can be used together in cells. As Nano-lanterns are not subject to photobleaching...phototoxicity, they successfully imaged multiple subcellular compartments over several minutes using Nano-...concentrations of the compound can be added to the media. Cellular esterase then converts diacetyl coelenterazine-h...

...article was written by Alyssa Cecchetelli and Andrew Hempstead. What do smell, taste and sight have in common...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https://doi.org/10.1016/j.cell.2017.03.028 Roth BL (2016...to the seven alpha helices that transverse the cellular membrane. The receptor recognizes the appropriate...signaling is initiated when a ligand binds to the extracellular surface of the GPCR. This results in a conformational...small molecules to elicit a response in a certain cell type. Chemogenetics is similar to optogenetics but...

...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...