...structures based on morphology. Autophagosomes are relatively easy to identify: double-membraned structures containing...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive...connected by a 9 amino acid linker that’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of...are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott, M., ...traditional method for studying autophagy. This method relies on the identification of autophagic structures ...lysosome, since both Atg8p and LC3 are degraded or released once an autophagosome fuses with the vacuole or...

...activity on NAG PAMS and reduced activity on NGG PAMs relative to wild type. A new genome-wide CRISPR knockout...) Strategy for leak-free transcriptional control. C) Iterative transduction workflow overview. Image from...nucleases identified, functions at temperatures up to 43 °C and can be used for genome editing in moderate thermophiles...opportunities for investigating genetic mechanisms related to morphogenesis, cell fate specification, and ...the internal control (Fig 1). Because this method relies on the fluorescence ratio between the 2 fluorophores...

... amplification in the host cell. This enables relatively high and long-term expression of the reprogramming...the reprogramming factors- OCT3/4, SOX2, KLF4, and C-MYC [1]. For the derivation of clinical grade hiPSCs...vectors that encoded six factors - OCT3/4, SOX2, KLF4, C-MYC, LIN28 and SV40 large T antigen (SV40LT). The ...of TP53 enhances iPSC generation as does replacing C-MYC with L-MYC [10, 11]. While the mechanisms underlying...understood, the addition of an shRNA for P53 and replacing C-MYC with L-MYC in the episomal reprogramming cocktail...patients are reprogrammed less efficiently, no such correlation has been found between the age of the patients...Models for Disease & Open Science: Interview with Darrell Kotton Four Factors that Differentiate the Stem...

...fragments: N-terminal BirA* (N-BirA*) and C-terminal BiraA* (C-BirA*). These two fragments are fused to... are enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One ...also which fragment of BirA*, either the N- or the C-terminus, the bait proteins were tagged with. It’s...These interactions are often modeled as binary relationships, i.e. protein A interacts with protein B; but...transient PPIs. Many PPI identification techniques rely on protein interactors to stay in close contact ...

... with 40–60% G/C content are most likely to be successful, although sequences with G/C content outside...of the 3′ extension of the pegRNA should not be C. A C base is speculated to base pair with G81 of the..., J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A.,...Figure 3: pegRNAs whose 3′ extension begins with a C base can cause unwanted base pairing (left), while... Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). ... Knipping, F., Ravisankar, P., Chen, P.-F., Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, ..., Anzalone, A. V., An, M., Newby, G. A., Chen, J. C., Hsu, A., & Liu, D. R. (2022). Engineered pegRNAs...

...2018). For C>T and A>G base editors, the intended edit must be in a 5 - 10 nt window relative to the PAM...target sites that code for amino acids close to the C’ terminus of the protein, to maximize the chances ...bystander edits are possible if there is another target C or A in the window. Another technology, prime editing...doi.org/10.1038/s41587-020-0561-9 Dempster JM, Pacini C, Pantel S, Behan FM, Green T, Krill-Burger J, Beaver...JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang P-Y, Daley G, Church G (...introduction of a specific mutation, one generally relies on homology directed repair (HDR) to incorporate...along with the single cell clone (although this is rarely done). When targeting a dsDNA break for HDR, the...

...Time: ~40 minutes Equipment required: 37°C heat block and 62°C heat block Approach: Viral RNA is extracted...range Time: ~1 hour Equipment required: 37°C water bath and 42°C water bath Approach: RNA is extracted from...10.1038/s41596-019-0210-2 Lucia, C., Federico, P.-B., & Alejandra, G. C. (2020). An ultrasensitive, rapid...Valois, E., Solley, S. C., Braig, F., Lach, R. S., Baxter, N. J., Kosik, K. S., Arias, C., Acosta-Alvear, D...incubation  Time: ~90 minutes Equipment needed: 37°C heat block, LED blue light illuminator or UV light... in a range of temperatures varying between 10-50°C. Visit Addgene's COVID-19 Plasmids and Resources ...Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan C-Y, Guevara H, Wadford DA, Chen JS, Chiu CY (2020) ...

...While this method can be time-consuming, it is relatively effective in reducing off-target effects since...still unexpectedly affect nearby genes (Figure 1A-C). Find CRISPR plasmids at Addgene! Knock-in: Introducing...site (TSS) deletion. (B) Mutations of splice sites. (C) Removal of an exon or a large genomic fragment. (... doi:10.1080/15476286.2014.996067 Basak J, Nithin C. Targeting Non-Coding RNAs in Plants with the CRISPR-Cas...

... 25°C. At low temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies...genomes Compared to humans and mice, flies have a relatively simple genome. They have four chromosomes: the...females do not. Also, males have sex combs on their forelegs which are black, bristly hairs. Females do not...

...., 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., ...AdVs are ubiquitous (Schmitz et al., 1983), yet rarely cause severe disease in humans (and no vaccines...stages of infection for efficient cell lysis and release of adenovirus from infected cells. J Virol, 70(...

...Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus...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...

...jcm.23.5.959-961.1986 Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., & Berkmen, M. (2012... some people still use amp? Carb is expensive (relative to amp), and for some applications, the benefit...References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks, G. F. (1986). Effect...their mode of action and have a very similar relationship as amp and carb (they are interchangeable). ...low rate, there is evidence that zeocin is never entirely detoxified, even when Sh ble is expressed (Trastoy...

...T-regs) into their microenvironment by producing C-C motif chemokine 22 (CCL22) and TGFβ. T-regs are suppressive...tolerance-producing antigens enunciated above. Finally, signals released from the microenvironment help create tolerance...eradicate metastasis or eliminate the probability of a relapse. Chemical and radiation therapies cause a transient...

...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...FRET occurs. By photobleaching the acceptor, you release the donor's quenching and the fluorescence of the...

... 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... strides in making science more accessible and reliable. We continue to expand our educational content...

...Bryson, C., Fogg, M., Baker, M. P., Herskovitz, J., Goletz, T. J., Zhou, L., Moxness, M., Flynn, G. C., Narhi...trained on vast amounts of data to calculate the relative immunogenicity score (Mattei et.al., 2022).    ...Antidrug Antibody Formation in Oncology: Clinical Relevance and Challenges. The oncologist, 21(10), 1260–1268...

...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....serotype, as some rAAV serotypes are abundantly released into the culture media, while others remain cell-associated...Wilson, J. M. (2010). Efficient serotype-dependent release of functional vector into the culture medium during...

.... K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q.,...representative micrograph (C), particle orientation distribution and Fourier-shell correlation curve (D), and the... 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...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...

...changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G•C are mediated...called point mutations that change C to T, G to A, A to G, and T to C, represent about two-thirds of known...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464...Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR (2020...PACE and PANCE systems use M13 bacteriophage, which rely on the expression of gene III to create infectious...

...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3, that’s ...quenched in acidic environments, while mRFP is relatively stable and fluorescences even at the acidic pH... GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...autophagic flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...∆G, however, can not be conjugated to PE due to a C-terminal glycine deletion, so it remains in the cytosol...changes in autophagic activity, and are a more reliable indicator of autophagic activity. The aim of this...important to remember that because Keima’s fluorescent relies on lysosomal acidity, this biosensor can’t be used...