...find yourself defaulting to the most popular or familiar options without even realizing it. Yet, this selection...60 minutes of illumination, highlighting its reliability for long-term experiments (Salari et al., 2019...unconjugated dye molecules are critical for accurate and reliable imaging and assay results (Hayashi-Takanaka et...limit of light. Dyes like silicon-rhodamine or Janelia Fluor (JF) dyes have specific properties that position...biotechnology. Recent examples of advancements include the Janelia Fluor(R) (JF) dyes developed by the Lavis Lab (...interchangeable labeling using the HaloTag® system, the new Janelia Fluor HaloTag Ligands expand the self-labeling ...self-labeling capabilities of this platform. The Janelia Fluor ligands provide brighter and more stable fluorescence...

...epitope tags at target genes in mammalian cells (Bressan et al. 2017; Liang et al. 2017). Current methods...Cas9 protein-based method for epitope tagging in mammalian stem cells. For easy scalability to the 96-well...Ribonucleoprotein (csRNP) delivery Epitope tagging of mammalian genes using CRISPR depends on successful delivery... function of large protein cohorts in diverse mammalian cell types. A detailed step by step protocol for...enables rapid and precise genetic manipulation of mammalian neural stem cells. Development 144: 635-648. PubMed... and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein. Elife...Rev Genet 32: 601-618. PubMed PMID: 9928493. 4. Liang X, Potter J, Kumar S, Ravinder N, Chesnut JD. 2017...

...understand how to use it. TLDR: This is a versatile mammalian expression vector that you can use to express ...express both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses...that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component...backbone can be used to transfect a variety of mammalian cells and can be modified for viral trasduction...by western blot. Overall, this is a versatile mammalian expression vector that you can use to express ...SpCas9 and your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain...Some Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit...

...organisms with the flip of a switch. You may be familiar with popular light-sensitive ion channels used...OptoTIAM (magenta) expressed in a monolayer of endothelial cells, showing cell protrusions and overlaps ... Rab35 to understand their roles in forming the cilia that help establish left-right organization of the...The new SuperPLDs work with high efficiency in mammalian cells and in vitro, opening up a wide realm of...directed-evolution methods, the activity-based selection in mammalian cells used here could be an attractive strategy...2023). Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer ...scale, enabling precise control over vascular endothelial barrier strength. eLife, 12, RP84364. https:/...

...tag or a fusion protein. Mammalian vectors One of our most popular mammalian expression vectors, c-Flag...the pcDNA series. It has the CMV promoter for mammalian expression, a multiple cloning site to insert ...cassette for growth and cloning in bacteria and a mammalian selection cassette to screen for positive transfected...transiently transfected cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). .... You can also upgrade your basic mammalian expression system and go for a lentiviral or a retroviral ... Lianna Swanson...

...protocol link). We have found this method to be reliable and reproducible. However, we do not use amplicons...which does not a standard curve and somewhat more reliable quantitation. Differences in sample treatment ...any AAVs that have successful transduction in microglia? I have been using the AAV.PHP.eb for neuron, ...specificity and would like to get it working in microglia as well. A:  We are not aware of any available...available AAV for efficiently targeting microglia in vivo.   Q5: What is the efficiency of co-delivering different... capsid, particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver...might be worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently...

...for systemic gene transfer in primate brains Endothelial-specific AAV delivery Recombinant anti-FLAG tag... unleashes the secrets of primate brains with brilliance By Hridaya Patel (Addgene co-op) Researchers ...10.1038/s41565-023-01419-x. PMID: 37430038. Endothelial-specific AAV delivery By Cythina Arokiaraj (Gradinaru...to that of AAV1. This viral vector has brain endothelial cell-specific tropism, with slightly reduced ...proof-of-concept study, mouse LY6A was delivered to brain endothelial cells in mice lacking LY6A using AAV1-X1 (Chen...X1 and AAV9-X1.1 efficiently transduce brain endothelial cells across diverse mice strains and rats. Figure...across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism...

...express siRNAs of their choice in mammalian cells.  Learn more about Mammalian shRNA Tools for RNAi! Cre-Lox...control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown. Vector provided by the Qi lab....library to support functional investigations of the mammalian genome that can reveal how genetic alterations...introduction of multiple genetic alterations into mammalian cells. The MuLE system kit from the Frew lab consists...high throughput genetic modulation studies in mammalian cells and tissues. This modular and flexible system...construct complex lentiviruses for modification of mammalian cells with a single viral infection. References...

...of the PCR from 60° C to 61° C improved assay reliability. Different primers can have different annealing...that vary up to two-fold. How can we get more reliable AAV titering? For our viral service, we currently...vector preparations by qPCR. To ensure accurate and reliable results, we have optimized our assay in a few ...we ensure that the standard curve generated is reliable, and can thus be used to accurately infer titer...compare the values to determine accuracy and reliability. When doing this, we make a judgement call on...within the error of the assay and thus are deemed reliable, or if the sample needs to be quantified again...Central PMCID: PMC5445070. 6. Wang, Feng, et al. "A reliable and feasible qPCR strategy for titrating AAV vectors...

...specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300...Tet1-CD for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-...DNMT3A-PuroR for targeted cytosine methylation in mammalian cells. Co-expression markers EGFP and PuroR enable...vector in chick embryos, it also functions in mammalian expression systems.   Christopher Newgard’s Lab...Jaenisch R (2016) Editing DNA Methylation in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org... this publication at Addgene. Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein...5615–5628 . https://doi.org/10.1093/nar/gkw159 Williams RM, Senanayake U, Artibani M, Taylor G, Wells ...

...Research in Australia I then travelled abroad to work at the University of Queensland in Australia, this was...months in Australia or another country of the Commonwealth. Another reason why I chose Australia was because...because I knew the tests worked and would produce a reliable result. This is not to say the role was without...large scale data analysis, as this is increasingly reliant on programming skills which are not my strong point...downside to large commercial companies which are reliant on contracts with clients is that, when those contracts...

... the specific library. This protocol assumes familiarity with standard bacterial transformation and basic...

...biosafety level 2+. Please ensure that you are in compliance with your institution’s biosafety regulations...

... Thank You to Our Guest Blogger! Theresa Liao (@theresaliao) is the Communications Coordinator for the...This post was contributed by Theresa Liao of the University of British Columbia. When transitioning from...is a resume? How is it different from a CV? Get familiar with the general format of a resume and the different... that you have handled large data sets and are familiar with statistical analysis. If you have done fluorescent...help put together a grant application? Are you familiar with the publishing or patent application processes...

...neurons (-3mnx1/Hb9), pan neuronal (elavl3/HuC), microglial/macrophage (mpeg1.1), and astrocytic (gfap).This...cell types, such as principal excitatory cells and glia, have been successfully targeted via viral particles...constructs available at Addgene includes bacterial and mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs...method to increase CRISPR editing efficiency in mammalian cells. Their marker-free coselection, analogous... HF-Cas9 (NGG)  Very low off-target activity; mammalian expression vector Rees H, et al. Nature Communications...inhibit Spy Cas9 function in both bacteria and mammalian cells. These CRISPR inhibitors may provide a layer...reduce off-target gene editing. The bacterial and mammalian expression vectors for these CRISPR inhibitors...

..., immune, inflammatory, angiogenic vascular, endothelial, and stromal cells - a rich soup of life encircling...proteins, such as human growth factor, vascular endothelial growth factor, and interleukin-6 (Witkiewicz ...“friendly” cells in microenvironments! Take myoepithelial cells for instance. They retard tumor formation...H. Barsky and his team tested the influence myoepithelial cells had on the conversion of non-malignant...invasive and malignant forms and noticed that myoepithelial cells prevented progression to malignancy, concluding...15261139. Barsky, Sanford H., and Nina J. Karlin. "Myoepithelial cells: autocrine and paracrine suppressors of...

...p2A-ferritin-p2A-mCherry Mammalian Expression 74308 pcDNA3.0-Magneto2.0-p2A-mCherry Mammalian Expression 74334...yeast cells. Named after the brightly-coloured Australian parrot, the dual colour-emission biosensor Rosella...26837755   SEPTEMBER 2016 Zika virus plasmids Now avaliable through Addgene The Vaithi Arumugaswami lab at...Researchers screening protein-protein interactions in mammalian systems have many assays to choose from, however...two-hybrid assay are not optimal for studying native mammalian protein interactions, whereas others may be too... and quantify protein-protein interactions in mammalian systems. The DULIP system is comprised of Gateway-compatible...further work is needed to assess its function in mammalian cells or other animal models, but this technique...

...be taken in reliance on the information contained in this post and we disclaim all liability in respect...technology transfer, communications, medical writing, compliance, core facility management, collaboration management...higher education nonprofit entities related to or affiliated with institutions of higher education nonprofit...institutions of higher education, a related or affiliated nonprofit entity, or nonprofit research organizations... to).  Immigration does not want people to be affiliated with groups they deem not in line with democracy...research, clinical, regulatory, project management, alliance management, business development, finance, and...

...cost-effective and faster-growing than mammalian cells, but mammalian cells can perform human-like post-translational...antibodies conveniently allow for unlimited production, reliable expression, and easy distribution as DNA (Trimmer...can take place in bacterial, yeast, plant, or mammalian cells. Each of these systems has their advantages...bonds (Frenzel et. al., 2013). If you’re using a mammalian production system, cell lines typically used include... your plasmid(s), and then wait 7-14 days (in mammalian cells) to collect the cell culture supernatant...

...  *Worried about your budget? Email janeliafluor@janelia.hhmi.org to try out the JF dyes. Acknowledgements...guest blogger, Luke Lavis, a Group Leader at the Janelia Research Campus, Howard Hughes Medical Institute...synthetic dye, mauvine, was discovered in 1856 by William Perkin. His discovery set off a flurry of activity...permeability (Figure 2c). These cell-permeable Janelia Fluor (JF) dyes have excellent properties inside...Luke D. Lavis is currently a Group Leader at the Janelia Research Campus, Howard Hughes Medical Institute...