...imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent reporter of mechanical...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs.... E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of monoclonal...39406745. Optimizing mScarlet tags for live imaging in C. elegans By Alyssa Neuhaus Red fluorescent proteins...maturation rate limits its use in systems like the C. elegans embryo, where developmental processes happen...recently generated new constructs specifically for C. elegans (Cao et al., 2024). The team designed coding...mScarlet3 and mScarlet-I3 that were codon-optimized for C. elegans and included artificial introns to improve...

...., Pan, C. Y., Rizzo, K., Bertumen, J. B., Morales, C., Oluniyi, P. E., Nguyen, J., … Chiu, C. Y. (2023...36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval, A., Harris-McCoy, K. C., Hensley, S. E., Zhou, D., Lin..., Iparraguirre, A., Lopez-Camacho, C., Wherry, E. J., & Ertl, H. C. J. (2007). Adenoviral vectors persist...Borkenhagen, L. K., Fieldhouse, J. K., Seto, D., & Gray, G. C. (2019). Are adenoviruses zoonotic? A systematic review....org/10.1016/B978-0-12-801238-3.02523-X. Hofmann, C., Löser, P., Cichon, G., Arnold, W., Both, G. W., ...toward saying that host-virus interactions are an important part of the biology of adenoviruses (AdVs). AdVs... Although host-virus interactions likely seem important for any virus, we will see that they are very ...

...CyPet with C-terminal His tag SCFP3A Cyan Mammalian Express a gene of interest fused to the C-terminus...YPet with C-terminal His tag SYFP2 Yellow Mammalian Express a gene of interest fused to the C-terminus...Mammalian Express a gene of interest fused to the C-terminus of monomeric Cerulean mTurquoise2 Cyan ...Mammalian Express a gene of interest fused to the C-terminus of Amber mVenus N1 Yellow Mammalian Express...Mammalian Express a gene of interest fused to the C-terminus of monomeric Venus pCEP4YPet-MAMM Yellow...Mammalian Express a gene of interest fused to the C-terminus of LSSmOrange mRuby2 Red Mammalian Expresses... theory, is an easy technique, but it is very important to follow some simple rules to avoid common pitfalls...

... D. W., Kumaran, R., Rego, M., Schofield, C., … Laflamme, C. (2024). A consensus platform for antibody... H., Laflamme, C., Houston, D. W., Polinski, N. K., Meijers, R., Levey, A. I., Saper, C. B., Errington...Ayoubi, R., Ryan, J., Gonzalez Bolivar, S., Alende, C., Ruiz Moleon, V., Fotouhi, M., Alqazzaz, M., Southern...We continue to expand our educational content supporting the tools in our collection, such as blogs describing...viral vector serotypes, or hundreds of Data Hub reports detailing antibody experimental results. We also...exactly what you need, faster. We’re also building a portal to easily share sequences and characterization ...

...specific genomic locus: C-BERST Article contributed by Klaus Wanisch  Listen to the C-BERST podcast segment...Zhao C, et al. RNA. 2018 PubMed PMID:29109157. Nanobody toolkit to monitor retrograde transport at the...Sontheimer lab used this technology, which they call C-BERST (dCas9–APEX2 biotinylation at genomic elements...in future studies of other genomic loci. Find the C-BERST plasmids here. Gao at al. Nature Methods (... RNA templates to DNA but suffer from several important shortcomings including low processivity (ie. the...functionalized nanobodies to study retrograde transport from the cell surface. Their toolkit includes ...

....   C. roseus is not a model organism such as Arabidopsis or tobacco. Studying gene function in C. roseus...De Ruijter, N. C. A., et al. "Evaluation and comparison of the GUS, LUC and GFP reporter system for gene...in tissue culture. This method is commonly used in C. roseus, but processes specific to photosynthetic ...Unver and Budak, 2009). This method works well in C. roseus. A common method for overexpressing genes ...of interest is transferred into the cells. But for C. roseus, Agrobacterium infiltration into leaves does...infiltrate. We have recently overcome this problem in C. roseus by working with young seedlings and optimizing... transformation. Choose good reporter genes There are many great reporter genes available and which one...

...an ortholog in C. elegans making it ideal to study basic molecular biology processes. C. elegans are also...PMCID: PMC5525645. Mello, Craig C., et al. "Efficient gene transfer in C. elegans: extrachromosomal maintenance...lab to study neuronal development (Brenner, 1973). C. elegans are small, transparent worms that have a ...life cycle and large brood sizes. The majority of C. elegans are also self-fertile hermaphrodites making... Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout their...GCaMP6 (Nguyen et al., 2015). If you are interested in C. elegans plasmids and resources check out Addgene’...cerevisiae to human: The important gene co‑expression modules." Biomedical Reports 7.2 (2017): 153-158. PubMed...

...Michael, S., Kuleshova, E., Ojala, D., Huang, C.-C., Gerfen, C. R., Schiller, J., Dudman, J. T., Hantman,...doi.org/10.1089/hum.2008.123 Dalkara, D., Byrne, L. C., Klimczak, R. R., Visel, M., Yin, L., Merigan, W....scitranslmed.3005708 Goertsen, D., Goeden, N., Flytzanis, N. C., & Gradinaru, V. (2022). Targeting the lung epithelium...Ishikawa, Y., Jae, L. T., Wosen, J. E., Nagamine, C. M., Chapman, M. S., & Carette, J. E. (2016). An essential... S., Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A...Horwitz, N., Beggs, A. H., Wagers, A. J., & Sabeti, P. C. (2021). Directed evolution of a family of AAV capsid... T., Rehman, K. K., Bertera, S., Zhang, J., Chen, C., Papworth, G., Watkins, S., Trucco, M., Robbins, ...

..., it is important to not overgrow the plasmid at 37°C. Some researchers find that using a 30°C incubation...pnas.77.8.4847 Trepotec Z, Geiger J, Plank C, Aneja MK, Rudolph C (2019) Segmented poly(A) tails significantly...https://doi.org/10.2144/000112585   Ding T, Huang C, Liang Z, Ma X, Wang N, Huo Y-X (2020) Reversed paired-gRNA...Working on the scientific support team at Addgene, I get to see everything that can go wrong with a plasmid...

...S., Croft, C. L., Futch, H. S., Ryu, D., Ceballos-Diaz, C., Liu, X., Paterno, G., Mejia, C., Deng, D.,...References  Aurnhammer, C., Haase, M., Muether, N., Hausl, M., Rauschhuber, C., Huber, I., Nitschko, H...Esbin, M. N., Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using...., Chtarto, A., Clark, K. R., Conlon, T., Darmon, C., Doria, M., Douar, A., Flotte, T. R., Francis, J....different titers are measured. A TCID50 measurement reports an infectious titer, meaning the concentration ...

...terminal); 2xNLS (C terminal) 47327 pET-28b-Cas9-His 6xHis (C terminal); NLS (C terminal) 62933...6xHis 6xHis (C terminal); NLS (C terminal) 62934 pET-NLS-Cas9-6xHis 6xHis (C terminal); NLS ...also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical mouse...the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR. Potential guide... homology arms and a selection marker, then electroporate it into mouse embryonic stem cells. After selecting...compatible with standard microinjection and electroporation methods using in mouse transgenics. Designing...between the ampulla and infundibulum. In vivo electroporation is performed immediately after injection. Image...

... standard strain, like DH5α, grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of ...freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. If troubleshooting, the safest...ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus may appear as a...freeze-thaw cycles on hepatitis B virus DNA and hepatitis C virus RNA quantification as measured with branched-DNA...that infect bacteria), many of which are doing important work in our microbiomes. Given that viruses are... and the type of virus, there have been varied reports of titer losses from 5% to 50% per freeze thaw ...vector: optimization of conditions for storage, transport and delivery." Japanese journal of cancer research...

.... K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q.,... module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected with...Protein samples protected from AWI by helical LEAs. C-E) cryo-EM single-particle analysis of PRC2 with RvLEAMshort...glutaraldehyde crosslinking: representative micrograph (C), particle orientation distribution and Fourier-shell...tardigrades!  Find RvLEAMshort here!    Abe, K. M., & Lim, C. J. (2024). Small LEA proteins as an effective air-water...-02078-y.  Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy...Serack, F., Harvey, J. W., Brauer, P. P., Pirtle, C. P., Coffey, A., Howard, M., Kamath, N., … & Weissman...

...defined between a fairly nippy 20°C to a decidedly uncomfortable 45°C). Their more adventurous cousins ...of growth, say around 37°C, is a far cry from their native conditions of ~0°C, which drastically affects... But what about placing the mesophilic hosts at 0°C? That won’t work out either - most mesophilic systems...severely cold conditions. To understand their unique importance, let’s begin with a few basics.  Mesophiles vs...understanding and appreciating their biology. The importance of psychrophilic enzymes From an industrial perspective...decided to create a hybrid system where we could incorporate cold-tolerance inducing genes within a regular...scientist from Addgene. The talk highlighted the importance of reagent repositories and the myriad facilities...

...into the A2 noradrenergic area of the caudal NST. (A-C) Low-magnification images showing viral spread. (D-F...Backer, M. W. A., Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization...Gruntman, A. M., Su, L., Su, Q., Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility...data free. Nature, 5–7. O’Connor, D. M., Lutomski, C., Jarrold, M. F., Boulis, N. M., & Donsante, A. (2019...Malaker, S. A., Poweleit, N., Maynard, L. H., Adams, C. M., Leib, R. D., Cirolia, G., Thomas, D., Stamnes...doi.org/10.1016/j.ymthe.2005.02.021 Zuk, P., Sanchez, C. E., Kostick, K., Torgerson, L., Muñoz, K. A., Hsu...experiments didn’t provide any information on other important factors such as what is the best promoter to use...

...110, 073704 (2017). Hurt, R. C. et al. Genomically mined acoustic reporter genes for real-time in vivo ...visible in cells via TEM, and purified GVs under TEM. (c) Ultrasound imaging of tumor colonization by bARGSer-expressing...general method to create your own mARGAna cell line. (c) Z-projected phase contrast images of GV expressing...What are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used... to study gene expression in cells. But these reporters have limited use in vivo because they are based... penetration depth as well as high spatial and temporal resolution, making it ideal for visualizing gene...expression in vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. ...

...construct for C. elegans gene knockdown Article contributed by Alyssa Cecchetelli  Listen to the C. elegans...segment RNA interference (RNAi) is extensively used in C. elegans research to study gene function. RNAi is ...commonly achieved by feeding E. coli expressing dsRNA to C. elegans. These dsRNAs target specific mRNA transcripts...previously not an option, including Drosophila and C. elegans.   Find the TurboID plasmids here! Branon...producing long unspecific RNA fragments that are incorporated into the dsRNA necessary for RNAi. The Vellai...adjacent to the T7 promoters to prevent the incorporation of non-specific nucleotides into the dsRNA leading...enzyme and a small-molecule substrate to map the spatiotemporal distribution and interaction networks of specific...

..., Banh, L. M., Williams, C., Toh, M. S., Irby, M. J., Edwards, L. S., Lin, C., Owen, A. L. G., Künne, ...contains all five of these Cas proteins, while type I-C Cascades do not contain Cas6. Regardless of the subtype...Resources References Loeff, L., Brouns, S. J., & Joo, C. (2018). Repetitive DNA reeling by the Cascade-Cas3...Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5....Coons, M. M., Klompe, S. E., Lied, A. M., Smith, S. C., Vidal, B., Donohoue, P. D., Rotstein, T., Kohrs,....com.   The Cascade complex Cascade is an important component of the CRISPR Cas3 system. Most Cascade... appears to be conserved. This DNA degradation portion can be bypassed by leaving out Cas3 — a tactic ...

...Park, J. C., Zhao, J., Luebbers, A., Green, E., Philibert, C. E., Zhang, H., Layne, M. D., Wu, J. C., & Garcia-Marcos...including "CLAP", combining an N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed...human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured rat hippocampal neuron expressing pan-membrane...the soma. The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted...all the components of the biosensor in optimal proportions from a vector backbone that allows lentiviral...Waldron We recently added two antibodies against important targets for neurodegeneration research: Anti-MAPT...6E10-A5] in Immunohistochemistry (Human). Addgene Report, doi: https://doi.org/10.57733/addgene.6ncijt.  ...

...PCR or by evaluating visual phenotypes. Improving C. elegans fluorescent protein tagging with SapTrap ...called SapTrap to improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one...protein tagging systems For those who do not work with C. elegans, several groups have designed modular toolkits...by the Hornung Lab, allows users to easily create C-terminal tag fusions of endogenous genes in human ...plasmid containing the homology arms and an N- or C-terminal tag. The two PCR reactions are then mixed...line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag, YFP... mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled...