...and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures GFP is limited in its...proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays a critical...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...ability to interact with protein partners within the cell. However, antibodies can have low affinity for these... generally bright, stable and well tolerated by cells. They can be used for protein localization, isolation...trigger protein aggregation which is toxic for the cells and affects the labeling of small structures like...team, an ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with...

... flanking sequences to their input string (Figure 2). This string can then be used as input for the prediction...top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...Biotechnology. https://doi.org/10.1038/s41587-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, ... 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... balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/J.MOLCEL.2021.03.032 Yu, G., ...

...channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to easily detect opsin-expressing cells. The resulting.... "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit ...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents... specific cell-type promoter. Once injected into the brain, the viruses infect all the cells with an inactive...energy conversion or the induction of intra- or intercellular signaling pathways (Ernst et al., 2014). Microbial...proteins control the flow of electric charges across cell membranes and maintain membrane potential in response...

...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and...be challenging – is advice and guidance from only 2 people enough?  For many years I’ve been organizing...Apparently, a good Jedi does not necessarily an excellent mentor make. Are your current mentors/advisors...What Kind of Mentor Do I Want To Be?” In this excellent essay Acclimatrix, an assistant professor at a...

...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...not generally in a cell’s best interest to overexpress a protein. Energy and cellular resources are being...being spent to make something the cell doesn’t need to make. Eukaryotes and some bacteria deploy proteosomes...proteosomes to degrade what the cell might consider junk protein. Though there are a number of chemical and...same manner as it would have in the context of a cell. FLAG tags can be cleaved by enterokinase, which...

...untagged expression of SARS-CoV-2 proteins in bacteria, yeast, and mammalian cells. We teamed up with Ginkgo...approximately 3,000 constructs to support SARS-CoV-2 research.  Browse the entire Ginkgo Bioworks COVID...organizations to share materials more broadly to accelerate COVID-19 research: Sinai Health System                                     ...

...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure in ...ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure on Twitter... remember what happened when drug X was added to cell line Y, or the phenotype of a mouse with a specific...comparison of the same experiment from different cell lines/model systems ReFigure allows you to make...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor... that your reference genes are suitable for your cell lines and/or tissue types.  Please do note my oh-so-subtle...reference genes to save on plate space. For many cell lines and tissue types, appropriate reference genes...

...Epub 2016 May 2. PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan... be stable in the cell. gDNA guides will also need to be transfected into the cells of interest. In contrast...unable to show TtAgo-mediated cleavage in mammalian cells, likely due to high temperature requirements. To...plasmid and genomic DNA in a variety of mammalian cell lines, with an efficiency similar to that of Cas9...

...you look at the dot plot in Figure 2, you might assume that all cell populations are positive for both ... the case of Figure 2, one population is B- and another two are B+. Our A-B+ cells are in the lower right...right corner.      Figure 2: This flow plot has two populations that are B+ and one population...-B+ cells, and A+B+ cells, but no cells that are A+B-.      Figure 3: It's often easier to identify...population of B cells and dendritic cells (DCs) in the lower middle right. DCs and B cells are HLA-DR+ and...single cells in a population. It can be used to measure protein expression, identify rare cells, or even...looking for cells in a sample that are negative for marker A and positive for marker B, or A-B+ cells. If you...

... Plant Methods. BioMed Central, 2(1), p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829. PubMed...PMID: 24032037. PubMed Central PMCID: PMC3759027. 2 Harrison, S. J. et al. (2006) ‘A rapid and robust ...incubated at stationary phase for too long, the cells in your culture will no longer be viable. The time...graduate student at Stanford University. She studies cell fate maintenance in the stomatal lineage and has...

...starting day 2 after transfection.     Fig: 2: Expressing mARGAna in MDA-MB-231 cells. (a) Plasmid...expression. When engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase...producing cells. After FACS, return the cells to doxycycline-free media and expand the cells. Use a subset...white inclusions in cells with GVs via phase contrast microscopy, GVs visible in cells via TEM, and purified... heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...expression homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle...transient transfection in rtTA+ cell lines (such as HEK293-TETOn cells), or they can also be integrated...

...daughter cell, instead of partitioning each type of plasmid into a separate daughter cells (Figure 2) (Ebersbach...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet...copy number in the cell is high, but allows replication when copy number in the cell falls too low (Novcik...system to partition themselves to each daughter cell during cell division. They don’t simply rely on chance...eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When...from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher...

...vector:insert ratio is used in the DpnI digestion and 2 µL of digest product are used in the transformation...Biology 38 (2016): 145-154. PubMed PMID: 27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified...properly. Directly transform chemically competent cells with the digested mixture. There is no need for ...purification before transformation. After transforming cells, you should perform colony PCR with vector specific...interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis...

...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...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...only 1,700 genes total). Hydra (Celina Juliano) This is a known stem cell and regeneration model. Little...Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to study for convergent evolution because...

...into the several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, ...mounting sample on slides, and confocal   Figure 2: High-level overview of an indirect immunofluorescence...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...

...Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Find...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify... adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable A to I...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . https://doi.org/10.1038...specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869–874 . https://doi.org/10.1038...

...native enzyme, which are summarized in Table 2 below.  Table 2: Properties of native luciferases Organism...luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and deliver ...miRNAs for effectiveness. Analyze Cell Signaling Pathways Known cellular signaling pathways can be investigated...luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays have since become a gold standard... serves as an indicator of transcription within cells, where detection of the reporter protein or its .../Phototoxicity Not susceptible Susceptible Subcellular Imaging Improving Well-established High-throughput... into the desired mammalian, plant or bacterial cells. Expression of luciferase as the reporter gene is...

...the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind...PMC2746483 Find ribosome profiling plasmids at Addgene. 2. Halstead, James M., et al. “Translation. An RNA biosensor... of all of the mRNA bound-ribosomes in a cell. When cellular mRNAs are digested with ribonucleases, ribosomes...Regulating translation is key to cellular function, especially during development or stress. With ribosome...explore the complexity of translational control in cellular physiology. Ribosome profiling Ribosome profiling...the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing that ~80% of transcripts...powerful tools for studying translation in various cell types and physiological states. References 1. Ingolia...

...Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells’ nuclei--our chromatin is comprised...target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence...plant protein TIR1 in non-plant cells, which interacts with the cells’ endogenous ubiquitin ligase, the...translate AID to yeast, as well as mammalian and human cell lines (Nishimura et al., 2009, Natsume et al., 2016...based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, the lab was able to... protein of interest to create conditional human cell lines. Lastly, the Kanemaki lab demonstrated that...the target sites adjacent to an NG PAM in human cells. The increased versatility of the engineered SpCas9...