...   References and Resources   References Beck C, Zhang D, Gong Y. Enhanced genetically encoded voltage...: 10.1016/j.cobme.2019.10.010. Bando, Y., Grimm, C., Cornejo, V.H. et al. Genetic voltage indicators.... 71 (2019). doi:10.1186/s12915-019-0682-0. Beck, C., Gong, Y. A high-speed, bright, red fluorescent voltage...sensors are better classified by their fluorescent reporter and the mechanism of their voltage sensing component...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...for penetrating deeper tissues depths (1 mm), important for certain applications such as in vivo tissue...rhodopsin Ace2N and mScarlet. Ace2N quenches a proportion of mScarlet's emission via FRET.  Image from ...

...doi.org/10.1038/mt.2009.280 Hirsch ML, Li C, Bellon I, Yin C, Chavala S, Pryadkina M, Richard I, Samulski....2010.205 Trapani I, Colella P, Sommella A, Iodice C, Cesi G, Simone S, Marrocco E, Rossi S, Giunti M, ...Trapani I, Toriello E, de Simone S, Colella P, Iodice C, Polishchuk EV, Sommella A, Colecchi L, Rossi S, Simonelli...1964 . https://doi.org/10.1172/jci34316 Bak RO, Porteus MH (2017) CRISPR-Mediated Integration of Large ...Large Gene Cassettes Using AAV Donor Vectors. Cell Reports 20:750–756 . https://doi.org/10.1016/j.celrep.2017.06.064...

... 25°C. At low temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies...combs. To make genetic crosses properly, it is important to isolate females from males while they are still... after hatching into their adult phase. It is important to clear out the parent crosses from the container...feature when setting up genetic crosses. More importantly, this knowledge will stop you from embarking ...

...Adenine base editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464...Lee S-J, Liquori AJ, Murray R, Packer MS, Rinaldi C, Slaymaker IM, Yen J, Young LE, Ciaramella G (2020...selection of guideRNAs. Of particular note, ABE8s reported in this work did not cause genome-wide, guide-...guide-independent off-target deamination, an important attribute for therapeutic application. ABE8s demonstrate...

...four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated somatic cell to...additional pluripotency stage) Less Safety Less (C-MYC, a known oncogene is one of the reprogramming ...22645348. PubMed Central PMCID: PMC3390875. 25. Patsch, C., et al., Generation of vascular endothelial and smooth... PubMed Central PMCID: PMC4363833. 35. Eguizabal, C., et al., Dedifferentiation, transdifferentiation,...1): p. 82-94. PubMed PMID: 23329641. 36. Jopling, C., S. Boue, and J.C. Izpisua Belmonte, Dedifferentiation... adenovirus [5], Sendai virus [6], plasmid electroporation (episomal) [7, 8] and mRNA transfection [9]...insulin-producing β-like-cells [37, 38]. In 1987, Davis et al. reported one of the earliest examples of transdifferentiation...

...assembly of NgAgo/ssDNA requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian...genome or just after a stop codon (as required for C-terminus epitope tagging). NgAgo survey Since it was..., he tries random things (101 lessons on Spanish, C++, guitar, photography) and often dreams of a sunny...Researchers from a variety of institutions recently reported their inability to recapitulate the results of...

... P., Rodriguez-Aguayo, C., Walton, B. L., Sood, A. K., Greaver, J., Janssen, C. F., & Lopez-Berestein,... Academic Press. Turner, P. V., Brabb, T., Pekow, C., & Vasbinder, M. A. (2011). Administration of substances...muscle groups (usually the thigh), though it is important to avoid injection into the sciatic nerve. Mice...

... should already be at 37 °C. Most labs have a water bath constantly at 37°C for this purpose.Learn about... Cells usually have their comfort zone around 37 °C and 5% CO2 and get agitated with sudden changes in...passage are too young and should not be used for important transfection experiments right after they’ve been...

...GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes ~15min, TagRFP ~100min and DsRed ~10hours...instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most suited for mammalian or bacteria...GFPS65T is better suited for yeast studies (24-30°C). Brightness: Brightness is a measure of how bright...temperature). pH Stability: This parameter is important if you are planning to express the FP in acidic...

...TurboID and APEX2 at different cellular locations. C) Enrichment of dual-labeled proteins identifies proteins...bioRxiv (A) and Qin and Cheah et al. 2023 bioRxiv (B-C). They were able to label proteins moving to and...fiber labeled with HaloTag-iPAK4. Scale bar 10 µm. C) Images of iPAK4 fibers in cultured rat hippocampal... editing efficiencies in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and...., 2023, reused under CC-BY 4.0 License. Importantly, even commercial antibodies can be tethered on... more. For added precision, the authors also incorporated LOV-Turbo into TransitID experiments.  Find ...in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells...

...References and Resources References Radtke, A. J., Chu, C. J., Yaniv, Z., Yao, L., Marr, J., Beuschel, R. T.... J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R., Yao, L., Yaniv...along with the ability for any user to upload supporting documentation that agrees or disagrees with the...

...site for a total of 2 binding sites per antibody. C) IgG’s are big enough that multiple labels (green ... IgG secondary antibody (Pleiner et al figures 4A-C). And since they have limited cross-reactivity between...Bates, M., Böhning, M.I., Chug, H., Görlich, D., Lee, C., Pleiner, T., Schliep, J.E., Stark, H., Trakhanov... genetic engineering of a tag or fusion with a reporter enzyme. For multicolor staining, careful consideration...al. Before picking a secondary nanobody, it’s important to review a few characteristics of your primary...

... Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA ...obtain even more precise temporal control of gene knockout or activation, the c-terminal cas9 fragment ... the nuclear export sequence. Treatment with rapamycin induces Cas9(N)-FRB-NES and Cas9(C)-FKBP-2xNLS ... (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This system provides users greater temporal control ...an n-terminal DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's group utilized...fragment was fused with FK 506 binding protein 12 (Cas9(C)-FKBP-2xNLS) and the n-terminal cas9 fragment with ...constructing fusion proteins with customizable N- and C-terminal modules   Thanks to the lab of Tomás Santalucía...

...References and resources References Feng, J., Zhang, C., Lischinsky, J. E., Jing, M., Zhou, J., Wang, H.,...j.neuron.2019.02.037 Kagiampaki, Z., Rohner, V., Kiss, C., Curreli, S., Dieter, A., Wilhelm, M., Harada, M....Neuromodulators like dopamine and norepinephrine have important functions in the brain but have been difficult... are blocked by alpha1-AR antagonist. This is important because alpha2-AR can provide inhibitory feedback...

...) 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...straightforward workflow for doing gene targeting, reporter generation, controllable gene knockouts, and inducible... models, toolkits like this one provide new opportunities for investigating genetic mechanisms related...

...constructed 22 different mTFP1 fusions to both the C- and N- termini of the fluorescent protein and found... PubMed Central PMCID: PMC2292683. Shaner, Nathan C., et al. "A bright monomeric green fluorescent protein...enhanced GFP (EGFP)-type termini. mNeonGreen was reported as the brightest monomeric green or yellow fluorescent... or for live cell imaging of fusions and FRET reporters. mTFP1 is also insensitive to physiologically ...

...the annealing temperature of the PCR from 60° C to 61° C improved assay reliability. Different primers... PubMed Central PMCID: PMC2957240. 5.Taylor, Sean C., Genevieve Laperriere, and Hugo Germain. "Droplet... people usually report? Addgene and other viral vector manufacturing facilities report the physical titer... characteristics give us information on three important factors. What can AAV titers tell you? Physical...cell transduction assays. Wild-type AAV2 has been reported to have a near-perfect physical-to-infectious ... However, for recombinant AAV2, the same study reported a physical-to-infectious particle ratio of 50:...(“full”) (Zeltner et al., 2010), while another reported that only 20% of recombinant AAV2 particles were...

...from this publication at Addgene. 2. Swarts, Daan C., et al.  “DNA-guided DNA interference by a prokaryotic...from this publication at Addgene. 3. Swarts, Daan C., et al. “Argonaute of the archaeon Pyrococcus furiosus...NgAgo In eukaryotes, Argonaute proteins play an important role in RNA interference, binding RNA guides to...

...window) C-terminal Anti-MECP2 (M6818) mouse antibody from Sigma (Link opens in a new window) C-terminal...localization signal (not essential for localization) the C - t erminal D omain (CTD) The most common missense... than mutations further downstream in the NID and C-terminus. A large number of mutations are known to...26944080 (Link opens in a new window) Alysson Muotri c.806delG 806delG G269Afs*20 M Fibroblasts & iPSC Unpublished... cluster in the MBD and NID demonstrating the importance of binding to methylated DNA and the recruitment...syndrome at the molecular level. They are also an important tool for developing therapeutic strategies to ...MECP2 locus Castaneus MECP2-NLuc-tdTomato mouse reporter cell line (Link opens in a new window) PMID: 35148843...

...website.   Bibliography  Ersing, I., Rego, M., Wang, C., Zhang, Y., DeMaio, K. H., Tillgren, M., Fava, A.... E., Matho, K. S., Cetin, A., Woodard, K., Cepko, C., Guerin, K. I., Rego, M. A., Ersing, I., Bachle, ...10.3389/fnana.2019.00093 McDade, J. R., Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations...from 2016–2023 by type of tool. Equally as important to what is being delivered is where it’s being...vectors not only available, but accessible. The supporting data and community engagement required to do ...lessons learned from this process have shaped and supported our growth and development. We’ve shared what ...

...editing window, for example all C’s will edit to T’s. Thus, it’s important that there are no “bystander ... single base pair edit. (Cytosine deaminases edit C-to-T and adenosine deaminases edit A-to-G.) In short...takes place on the free strand of DNA and converts a C to U without producing a double strand break. ... particular gene isn’t feasible. For example, temporary repression or knockdown allow you to study essential...

...10.1126/science.1206593 Powers S, Miranda C, Dennys-Rivers C, Huffenberger A, Braun L, Rinaldi F, Wein....2020.107878 Sinnett SE, Hector RD, Gadalla KKE, Heindel C, Chen D, Zaric V, Bailey MES, Cobb SR, Gray SJ (2017... More recently, the Bird lab found that only a portion of the MECP2 protein is necessary to rescue the...Neurological Disorder by Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878...

... by isolating a calcium-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish...calcium indicators can be used in mouse, Drosophila, C. elegans, and zebrafish – essentially all of the standard...said. “We know CREB is really important, but where is it really important and when? That is not known.”...calcium-binding sites as possible for added sensitivity. As reported in Nature Methods, the FRET changes of these Twitch...

... GCR, GR, GRL nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR3C2 Plasmids... MR, NR3C2VIT nuclear receptor subfamily 3, group C, member 2 NR4A1 Plasmids NR4A1, GFRP1, HMR, N10, NAK... RZR-GAMMA, RZRG, TOR RAR-related orphan receptor C RXRA Plasmids RXRA, NR2B1 retinoid X receptor, alpha...transcription factors modulated by ligands. Two of the important regions of nuclear receptors are: 1) a ligand ...

... Cre: Cre recombinase is split in half into N and C terminal Cre fragments (NCre and CCre, respectively...Bacterial Ekker 47551 pDD104 (Peft-3::Cre) Cre eft-3 C. elegans Goldstein 48201 CAG-GFP-IRES-CRE Cre and ...system CAG Mammalian Pelczar 51268 pCAG-Co-InCreC C-terminal component of the Co-InCre system CAG Mammalian...GFP (CRE-DOG) EF-1 alpha AAV Cepko 69571 pAAV-EF1a-C-CreintG Cre recombinase dependent on GFP (CRE-DOG)...dependent on GFP (CRE-DOG) CAG Mammalian Cepko 69573 pCAG-C-CreintG Cre recombinase dependent on GFP (CRE-DOG)...-Cre Cre CMV RABV Jessell 73717 pMLS328 Cre eft-3 C. elegans Jorgensen 75267 pAAV-CIBN-CreC photo-activatable...split-Cre CMV Mammalian Hirrlinger 106369 pCMV-Tag3B-CCre C-terminal split-Cre CMV Mammalian Hirrlinger 106370...

...nitrogen or a dry ice/ethanol bath, and store at -80° C. Once thawed, the AAV aliquot should be used quickly...PubMed Central PMCID: PMC190186. 9. Grieger, Joshua C., and Richard J. Samulski. "Packaging capacity of ...Axonal Transport It has been demonstrated that certain serotypes can undergo axoplasmic transport. These...of neurons (retrograde transport) or towards the synapse (anterograde transport), which is a possible ...retrograde and anterograde transport, but they also have similar transport characteristics (velocities...strong CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...and using AAVs have in common? The three most important items when looking for a house and choosing an...

...mEosFP’s chromophore requires a temperature below 30°C and this is not ideal for experiments in mammalian...can form it’s chromophore at a more comfortable 37°C (5). Dendra2 photoconverts from green to red fluorescence... pH stability, chromophore maturation rate at 37 °C, turnover rate, and the level of background fluorescence...: 1035-1040. PubMed PMID:18724362. 8.Stiel, Andre C., et al. "Generation of monomeric reversibly switchable...impossible to determine specific protein turnover or temporal expression using standard FP-chimeric proteins...second. The first successful irreversible PA-FP reported was PA-GFP. PA-GFP was derived from Jellyfish ...localization, or leading to protein aggregation. Another important parameter to consider is the contrast ratio- a...

...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) ...Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung... https://doi.org/10.1126/science.aaq0179 Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC... to cleave and activate a reporter. The detectable signal from this reporter can be measured and quantified...and a Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect ...method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2) (Gootenberg...

...create solution C. And if a conflict resolver does their job well, not only will solution C be better than...many sources of conflict are substantive (“Person C told Person A one thing and inadvertently told Person... is customer support, but there are eight other teams who still retain a customer support element amongst... your technical skills, can open new career opportunities for you, if you can identify them in yourself...explaining my approach to conflict resolution, it’s important to recognize that there are different types of...or goal. Resolving these types of conflicts is important for the individuals involved, and also for the...responsibilities. Knowing which aspects of customer support each team “owns” is critical for day-to-day operations...

...LRRK2 C-His LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40376 pBACgus/LRRK2 1867-2141 C-His...2161 C-His LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40378 pBACgus/LRRK2 1867-2176 C-His...2161 C-His_T2031E LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40381 pBACgus/LRRK2 C-His_G2019S...2161 C-His_G2019S LRRK2 His polH Parkinson's Michael J Fox Foundation MJFF 40383 pBACgus/LRRK2 C-His_C2024S...mCherry-MAPTau-C-10 MAPT mCherry CMV Parkinson's, FTD Michael Davidson 55311 mTagBFP2-MAPTau-C-10 MAPT mTagBFP2... mKO2-MAPTau-C-10 MAPT mKO2 CMV Parkinson's, FTD Michael Davidson 58112 tdTomato-MAPTau-C-10 MAPT tdTomato... CMV Parkinson's Mark Cookson 13314 pCMVTNT PINK1 C-myc PINK1 Myc CMV Parkinson's Mark Cookson 13315 pcDNA-DEST53...