... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey...nucleotide-based ligand binders would prove to be highly adaptable tools. The key to aptamers’ flexibility is their...

...SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation tag (dTAG) system functions...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...leading to ubiquitination and proteasomal degradation. Adapted from Nabet et al., Chemical Biology, 2018...of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons are destabilizing...various enzymes and sent to the proteasome for degradation. The tags we will discuss here are all conditional...conditional, meaning that they only facilitate degradation of their fused protein under certain conditions...are reversible mechanisms of control. Protein degradation can be induced by the addition of a small molecule...

...NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites... in the promoter region of gamma hemoglobin (HGB1/2) leads to continuous expression of fetal hemoglobin...evolved TadA domain. Because ABE7.10 contains a heterodimeric fusion of wild-type TadA and TadA7.10, they...wild-type TadA and the evolved TadA region. The ABE8.x-m version contains only the evolved TadA and is ~...3 components:  a deaminase (TadA, originally from E.coli, named TadA7.10 in ABE7.10)  a catalytically...nickase)  a guide RNA that targets the complex of TadA and dCas to the genomic DNA of interest  ...When ABEs are in action they bind DNA so that the TadA domain can edit the short single-stranded DNA stretch...

...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...cell differentiation (a cell type important for adaptive immune responses, which flies do not have!) (4...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...provided by the major companies selling PCR reagents. Nowadays commercial polymerases are very efficient, but...

...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 ...studied in 40 labs as a great model for plant adaptation and speciation. Their phenotypes are diverse ...captivity. Sticklebacks demonstrate unusual parallel adaptation to disparate environments so can be used study...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...ubiquitination substrate Ikaros (IKZF1ZF2) (B). Image adapted from Kroupova et al., 2024 under CC BY license....design and studies of protein ubiquitination and degradation.   Find the CRBNmidi plasmid here!   Kroupova...

...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...Widrick, J. J., Messemer, K. A., Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A.... a table listing some of critical differences (adapted from Zhou and Melton, 2008, [43]): Characteristic...

...Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W., Arbab, M...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101...J. M., Xu, A., Sisley, T. A., Weissman, J. S., Adamson, B., & Liu, D. R. (2021). Efficient C•G-to-G•C ...

...and in the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...nuclear (MALAT1) and cytoplasmic (CKB1) RNA target. Adapted from Figure 3 Colognori et al 2022.   Find CRISPR...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...features and evolved PAM sites, making Cas9 an adaptable choice for many genome engineering endeavors. ...target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed up to multiple...   Choosing a Cas While Cas9 may be the most adaptable Cas currently available, the other Cas proteins...

... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...in the bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to ...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ... issues that can arise in this step: protein degradation, incomplete lysing/insoluble proteins, and protein...protein aggregation. Protein degradation, which would result in low protein yield, can be reduced by adding...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...triggers a cascade of events that results in the degradation of the target mRNA and silences its expression...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone... on human artificial chromosomes. Montreal, QC, Canada 45.5017°N 73.5673°W – Vanessa Sung Hundreds of...de-funding of crucial environmental research programs, Canada has been here before.  These attacks on science...Research Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy...Intellectual Ventures Laboratory. Vancouver, BC Canada 49.2827°N 123.1207°W – Karissa Milbury I attended...

...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...activation/repression. dCas9 has also previously been adapted to fluorescently label a sequence matching its ...

...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...the figures of interest along with the article metadata, legend and user created notes on a “Pinterest...

... fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil...immune system. The techniques used here could be adapted to studying other systems in which there is large... revealing the cellular behaviors that mediate adaptive and innate immunity in diverse tissue environments... to better understand T cell initiation in the adaptive immune response. It was known that T cells were...scale when using photoactivatable FPs - their degradation within labelled cells limits the timespan available...