...example gene targeted for tagging with GFP at the C terminus, resulting in one edited allele, or gene ... gene of interest and decide which terminus, N or C, to tag and which short amino acid linker to use. ...study the protein or structure of interest. For a C-terminal tag, as shown in the example above, we inserted...precisely where we need it, in this example, at the C-terminus of the gene of interest. To get the molecular...tagging mammalian cell lines, Drosophila cells, and C. elegans. In addition to generating fluorescent protein...HDR as the double-strand break repair pathway, relying instead on microhomology-mediated end-joining (...

.... W., Déjean, V., Dodson, C. A., Wallace, M. I., Mackenzie, S. R., Timmel, C. R., & Hore, P. J. (2016)...Polali, S., Anderson, E. D., Bell, A. M., Tzouanas, C. N., Avants, B. W., & Robinson, J. T. (2019). Magnetic...doi.org/10.1021/jacs.3c01076.  Wheeler, M. A., Smith, C. J., Ottolini, M., Barker, B. S., Purohit, A. M., ...Beenhakker, M. P., Kucenas, S., Patel, M. K., Deppmann, C. D., & Güler, A. D. (2016). Genetically targeted magnetic...Fusing EGFP to the self-labeling FlavinTag made a reliable one-component magnetoresponsive system that could...Chemical amplification of magnetic field effects relevant to avian magnetoreception. Nature Chemistry, 8...

...expressing iGluSnFR3, iGluSnFR4f, and iGluSnFR4s. C) Spike-triggered averages for single action potential-evoked...tagging: Choose untagged or add a versatile N- or C-terminal HA tag. Selectable and stable: A puromycin...integrase-based approaches. Find STITCHR plasmids here! Fell, C. W., Villiger, L., Lim, J., Hiraizumi, M., … Abudayyeh... Cenik, B. K., Aoi, Y., Iwanaszko, M., Howard, B. C., Morgan, M. A., Andersen, G. D., Bartom, E. T., &.../doi.org/10.1016/j.molcel.2024.11.007. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina...and PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing...only a small number of glutamate molecules are released in each synaptic event and they are rapidly taken...

...Yamada, K., Kuznetsov, I. A., Rand, E. A., Ochs, C. C., Jaber, Z., Gardner, K. H., & Chow, B. Y. (2022...eLife.84364.  Meiring, J. C. M., Grigoriev, I., Nijenhuis, W., Kapitein, L. C., & Akhmanova, A. (2022)...., N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Silva, J. D., Freshour, J., & Hehnly...j.ceb.2015.08.005.  Tei, R., Bagde, S. R., Fromme, J. C., & Baskin, J. M. (2023). Activity-based directed ...roles played by microtubules, Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs...

...doi.org/10.3390/v12050513 Hsieh C-L, Goldsmith JA, Schaub JM, DiVenere AM, Kuo H-C, Javanmardi K, Le KC, Wrapp...plasmids used for this study at Addgene which include a C-terminal deletion in the spike protein to improve ...Zengel JR, Sathyan V, Nguyenla X, Van Dis E, Catamura C, Yamashiro LH,  Begeman A, Stark JC, Shon DJ, Fox ...Wrapp D, Lee AG, Liu Y, Chou C-W, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang...received over 13,000 plasmids requests for COVID-19 related plasmids. These plasmids have reached labs in over...

...cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR recombination with...flexibility since I can store the cell lysates at -80 °C for up to 6 months before purifying the AAV (Choi ...in one long day, or the virus can be stored at 4 °C and buffer exchanged the next day. Refer to the AAV...production and purification! References 1. Aurnhammer, C., Haase, M., Muether, N., Hausl, M.A., Rauschhuber...four times in a 95% ethanol and dry ice bath to release the virus particles from the cells. Cell lysates...

...PMC3725456. Schwarz H, Zhang Y, Zhan C, Malm M, Field R, Turner R, Sellick C, Varley P, Rockberg J, Chotteau...aliquot of our recombinant antibodies is stored at 37 °C for a period of two weeks. After this time, quality...33151027; PMCID: PMC7665837. L'Abbé D, Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression...Sederstrom D, Steinmann D, Mozziconacci O, Schöneich C, Freund E, Ricci MS, Carpenter JF, Lengsfeld CS. Do...antibodies conveniently allow for unlimited production, reliable expression, and easy distribution as DNA (Trimmer...

...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the ... prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way both ...set up, conducted and analysed with care to get reliable information. Although two-hybrid systems are a...

...Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S et al. 2017. ...available for human and mouse genomes only and deals with C-terminus epitope tagging. If you work with other model...your crRNAs using other web-based design tools (for C-terminus tagging, pick a crRNA that cuts 8-15 bp downstream...insertion site (within ~8-15 bp from the stop codon for C-terminus tagging) GC content of the crRNA (aim for... genotyping can tell if HDR completely failed. If C-terminus tagging potentially disrupts protein function... O'Duibhir E, Quinn N, Tyrer A, Leitner MC, Plumb C, Kalantzaki M et al. 2018. An efficient and scalable...epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed repair (HDR) to...

...rise of literary giants such as Isaac Asimov, Arthur C. Clarke, James Blish, and John W. Campbell. This period...combination of the four bases of DNA – A, T, G, and C. In this movie Ethan Hawke’s character revolts against...editors (CBE) use a cytidine deaminase to introduce a C→T change at precise locations in the genome. This ...JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR (2019) Search-and-replace...Correction: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 559:E8...420–424 . https://doi.org/10.1038/nature17946 Lenay C (2000) Hugo De Vries: from the theory of intracellular...for dinosaurs wreaking havoc in a theme park.  Released in 1997, GATTACA is an American SciFi film starring...

...02708-5 Hrit J, Goodrich L, Li C, et al. (2018) OGT binds a conserved C-terminal domain of TET1 to regulate...89–92. https://doi.org/10.1038/301089a0. Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking...https://doi.org/10.7554/eLife.34870.  Kaas GA, Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR...   Epigenetics nation stuck with methylation correlation  In the 1980s, a lot of things were cool: Walkmans...problem is that many of these studies report correlations without clearly testing whether a certain methylation... dCas9-TET, allowing one to study the causal relationship between DNA demethylation and gene expression...

...alternating between 5 min at -20°C (or -80°C) followed by 5 min at 37°C (each repeated 5 times) works well...with any protein purification, but overnight at 30°C is a good start. Prepare a lysis solution containing... PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Exploring the...PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Development ...membranes will then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose...

... 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 ...

... when the genome editing frequency is low, the relative abundance of exon-skipped mRNA is high, likely...disorder of glycosylation, and Niemann-Pick disease type C. Although the three DMD studies referenced here represent... 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz...K, Cheng JK, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe...and large animal models with mutations in human-relevant exons 45-55, rather than in the traditional exon...

...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...

...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...

...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. ...

...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...

...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 ...