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... synaptic proteins Fluorescent protein system for C. elegans Fluorescent timers for studying cell cycle...native locus, but introducing large genetic changes in C. elegans with CRISPR can be difficult since it requires...can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach. Split-wrmScarlet...together, they fuse and fluoresce. The team engineered C. elegans cell lines to express wrmScarlet1-10 unattached...cell cycle. Cells that had IL-3 withdrawn became entirely red as they could not proliferate. Eastman et...human glycosyltransferase genes and other proteins related to cellular glycosylation. Get the library at ...

...labeling of the cell population. Figure 2: Dense (c), intermediate (d), and sparse (e) labeling of the...References: del Valle Rodríguez A, Didiano D, Desplan C (2011) Power tools for gene expression and clonal ...Feng Q, Zhou Y, Zeng S, Ren M, Jiang S, Ni H, Zhou C, Gong H, Luo M (2018) Cell-type-specific and projection-specific...these labor-intensive and invasive techniques to reliably label any post-mitotic cells in Drosophila. SPARC... be labeled in a given population is directly correlated to the length of the truncated attP site. The...

...particles or relatively short expression times, where as others, such as hSynapsin, are relatively weak and...this study were packaged at Addgene and had a relatively low recombination rate (0.1% and 0.13% respectively...neuronal phenotype in a mixed population (Burger, C. 2004, Aschauer, DF. 2013, O’Carroll, SJ. 2021). This...https://doi.org/10.1371/journal.pone.0076310 Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin... require gene expression in a specific cell type rely on transgenic models that express recombinases like...sites and the open reading frame is inverted in relation to the promoter, hence the name, double-floxed...homologous pairs, and that the degree of homology is correlated with the level of spontaneous recombination. ...

...Wilkinson P, van der Meer D, Dow D, Buser-Doepner C, Bertotti A, Trusolino L, Stronach EA, Saez-Rodriguez...-019-1103-9 Bialek JK, Dunay GA, Voges M, Schäfer C, Spohn M, Stucka R, Hauber J, Lange UC (2016) Targeted...Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann ...Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki..., Weir BA, Hahn WC, Marsh JA, Tsherniak A, Kadoch C (2018) Interrogation of Mammalian Protein Complex ...providing a new gRNA rather than engineering an entirely new protein. This makes it much cheaper to use...

...fragment of CIB1 and it was fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of ...fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714) halves, rendering...split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription of the...used the bacterial phytochrome BphS which produces c-di-GMP in response to far-red light (730 nm). This...VP64 which in turn transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional ... from A. sativa (Bubeck et al., 2018). LOV2 has a C-terminal helix that unfolds when stimulated by blue...Bietz A, Waldhauer MC, Börner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, Correia BE, Eils R, Niopek D (2018...

... the digestion of food, while brain tissue is relatively static). To operate both types of implanted ...it - the device has a rechargeable battery, is relatively small, and can be detached easily whenever necessary...Antonini, M.-J., Rosenfeld, D., Park, J., Garwood, I. C., Yan, W., Niroui, F., Fink, Y., Chandrakasan, A.,...monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes of interest, and stimulate...outputs for the system. Success! They also observed reliable optically evoked neuron activity for at least .... P. (2020). Modular Optoelectronic System for Wireless, Programmable Neuromodulation During Free Behavior... Multifunctional microelectronic fibers enable wireless modulation of gut and brain neural circuits. Nature...

...References and Resources References Braun, C. J., Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design...Sousa, A. A., Tsai, S. Q., Bengtsson, N. E., Lööv, C., Ingelsson, M., Chamberlain, J. S., Corey, D. P.,...M. J., Joung, J. K., Breakefield, X. O., Maguire, C. A., & György, B. (2019). High levels of AAV vector... activation, and knockout. Most CRISPR screens rely on lentiviral systems, a fact supported by the wide...still beats lentivirus. In mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo...not integrate into the host genome. AAV instead relies mainly on episomal expression. Episomes are circular...It is important to note, however, that while AAV rarely integrates into the genome, CRISPR may increase...

...4355596 King, S. R., Duggal, N. K., Ndongmo, C. B., Pacut, C., & Telesnitsky, A. (2008). Pseudodiploid Genome...34372494. Prussin, A. J., Garcia, E. B., & Marr, L. C. (2015). Total Virus and Bacteria Concentrations in...Xiao, H., Conway, J. A., Hunter, E., & Kappes, J. C. (1997). Functional RT and IN incorporated into HIV...tools in molecular biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses...After uncoating, reverse transcription follows the release of the two strands of viral RNA and of the viral...The genome of viral tools are often not virology related at all, and you will instead get expression of ...genome can direct the production, packaging, and release of progeny viral particles. And there you have ...

...cells. For example, Henry Lester’s lab optimized the C. elegans GluCl ion channel genes for expression in...Santana-Quintero LV, Katneni U, Simonyan V, Kimchi-Sarfaty C (2017) A new and updated resource for codon usage ...–34 . https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and ...doi.org/10.1093/nar/28.1.292 Sauna ZE, Kimchi-Sarfaty C (2011) Understanding the contribution of synonymous...usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind to and deliver...studies have shown that translational efficiency is correlated with codon bias across all endogenous genes in...you want to express may contain codons that are rarely used in the host you are using or come from an ...

.... Time Viral production in mammalian cells is relatively quick. After transfection, HEK-293T cells start...https://doi.org/10.1096/fj.01-0995fje. Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 ...doi.org/10.3389/fbioe.2021.796991. Urabe, M., Ding, C., & Kotin, R.M. (2002). Insect cells as a factory ... cells, and its lifecycle ends in cell lysis, releasing the virus to the supernatant. The virus will reach...

... use next-generation sequencing to measure the relative proportion of guide RNAs (gRNAs) across the population...our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA...commonly think of DNA as a linear polymer of A,T,G, and C nucleotides arranged in a double helix. People are...die or differentiate Step 3 reveals which NANOG-related genes are critical to stem cell survival. Knowing...

...and purification An affinity tag, generally a relatively small sequence of amino acids, is basically a...tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the affinity ... tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike other epitope tags –... Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used ...crystallizing it or testing its function. Small ubiquitin-related modifier (SUMO) can help with folding and stabilization...western blots, IP, co-IP, IF, flow -cytometry, but rarely used for purification as elution requires low pH...  0.8 Affinity and Purification Very small size, rarely affects function S-tag KETAAAKFERQHMDS  1.8 Solubility...

...both methods. These approaches have relatively low sensitivity, which can be problematic if you are trying...also influence antibody binding by altering the relative concentration of potential antibody binders and...little to no signal in the liver sample, which you do (C), so your antibody is validated! Genetic Strategies...10.1080/19420862.2015.1100787  Taussig MJ, Fonseca C, Trimmer JS (2018) Antibody validation: A view from...recommendations for improving antibody use and reliability (Roncador, et al., 2015; Taussig, et al., 2018...your target then you would expect to see good correlation between the levels of target detected in both... targeting a different epitope, and look for correlation between the results. Using antibodies against...

...expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore...codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes the histidine... ATG codon from an ACG codon using a base-editor (C>T conversion) they could induce the translation of...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464...re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency...2018). TLDR: The ACE reporter from the Harris Lab relies on the restoration of a frame-shift in mCherry ...quantifies APOBEC-Cas9 efficiency in real-time, it relies on DSBs which is not ideal for monitoring single...

...requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often...plasmids with different varieties of tags, both N- and C-terminal. Each set of plasmids uses the same restriction...

... Sellnow, R. C., Boye, S. L., Coberly, B., Bennett, A., Agbandje-McKenna, M., Sortwell, C. E., Hauswirth...Backer, M. W. A., Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization...Dudek, A. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Cheng, F., Qiu, J., Carette, J. E., & Vandenberghe..., J. Y., Han, J. H., Lee, H., Han, Y. E., Rah, J. C., & Park, H. (2020). Labeling dual presynaptic inputs... Porrot, F., Bohl, D., Marmé, F., Chérel, Y., Salvetti, A., Hurtrel, B., Heard, J.-M., Rivière, Y., & ...your specific experimental system, rather than relying on other systems as proxies.  Finally, the size...consider. First, the titer required is inversely related to transduction efficacy: the higher the efficiency...

...IgG2a 177559 Anti-c-Fos [N486/32R] c-Fos Human Mouse IgG2a 177560 Anti-c-Fos [N486/76R] c-Fos Human Mouse...IgG2a 177520 Anti-LRRK2/Dardarin, C-terminus [N241A/34R] LRRK2/Dardarin, C-terminus Human Mouse IgG2a 177521...glutamate receptor Mouse Mouse IgG2a 206619 Anti-c-Fos [N486/25R] c-Fos Human Mouse IgG2a 206620 Anti-Kv1.4 K...220411 LRRK2/Dardarin kinase, C-terminus [N241A/58R] LRRK2/Dardarin kinase, C-terminus Human Mouse IgG2a ...VAPA [N479/111R] VAPA Rat Mouse IgG2a 220414 c-Fos [N486/93R] c-Fos Human Mouse IgG2a 220415 Cavalpha2delta2...] N483/126 Rufy3 Mouse Mouse 182090 C-Fos scFv [N486/32] N486/32 C-Fos Human Mouse 182091 Mortalin/GRP75...84 scFv Rufy3 Mouse Mouse 190560 c-Fos scFv [N486/76] N486/76 scFv c-Fos Human Mouse 190561 BKbeta2 K+...

...Epitope tags are short peptides introduced at the N or C terminus of a protein that are bound by antibodies...enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the most common tags, FLAG...not at all. The introduction of a tag at the N or C terminus of a protein may disrupt the biological function...a specific application, or they exist but are unreliable for a variety of reasons (Baker, Nature). Tagging...

...please sign up here.        New toolkit for N or C terminal tagging of proteins for degradation using...has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins with mAID and other ...cytidine deaminase  Target-AID-NG, which mediated a C to T conversion in the target sites adjacent to an...Nureki’s lab engineered SpCas9 (SpCas9-NG) with relaxed preferences for the third base of the PAM so that...

...be 10 µg/mL. Pro-Tip Transducing too many cells relative to the number of virus particles reduces the transduction...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...well plate and left undisturbed for 13 days. (a, b, c) Colonies formed by expansion of single cells for ...antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or removed from ...