... References 1. J. R. Bateman, et al. Site-Specific Transformation of Drosophila via PhiC31 Integrase-Mediated... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...earlier, even genes not found in Drosophila can be studied. Using a Drosophila cell line, I’ve studied a transcription...described the fundamentals of how to work with Drosophila as an experimental model organism. I then described...your flies will be ready for experimentation. Drosophila are amenable to many different kinds of experimental...can be employed to look at oxygen consumption. Drosophila are also used in cancer research (3). The sky...

... activation system co-opted from yeast (1). It is a Drosophila geneticist’s main workhorse to turn genes...Duffy, GAL4 System in Drosophila : A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID...A Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow...he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression...second post in our quick guide to working with Drosophila, you’ll learn how to maniupate expression of ...be overexpressed or knocked down. To do this, Drosophila geneticists use the Gal4/UAS system. This incredibly...had nearly a decade of experience working with Drosophila as an experimental system. Jon enjoys studying...

...Recombination in Drosophila Learn about Cre-Lox Resources on Addgene.org Find Drosophila Plamids Drosophila CRISPR... ortholog in the Drosophila genome? Continue reading and I’ll show you how Drosophila can be used to push...favorite gene (YFG) in Drosophila (and even if there’s not!) the wealth of Drosophila genetic tools available..., and overexpress YFG in a Drosophila cell line. Knock downs in Drosophila cell lines are extremely effective...your research in new and exciting directions. Drosophila are very easy to manipulate genetically and have... second post will detail a major tool used by Drosophila geneticists (the Gal4/UAS system), and the third... how you can make your own mutant flies. Find Drosophila Resources at Addgene The birds and the bees ...

...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...absorbance is plotted on the y-axis. With the help of graphing software, you then generate a trendline for the...the quality of a standard curve scientists use graphing software to calculate the data’s coefficient of...

...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...from the thermophilic bacteria Geobacillus stearothermophilus (GeoCas9). G. stearothermophilus grows at...cells, yeast, drosophila, plants, worms, zebrafish, frogs, some bacteria; but not thermophilic (high heat...for facultative thermophiles (high OR medium heat loving), but not obligate thermophiles. However, the ...

...approximately 1 in 5000 males. DMD is caused primarily by frameshift mutations in dystrophin, a protein ... applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy...needed. First, CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout...also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular dystrophy...neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID...);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed PMID: 25830891. PubMed Central PMCID: PMC4393360...for proper muscle function. Without functioning dystrophin, an individual experiences progressive muscle...

...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...attR sequences for genome integration by phiC31) Drosophilia Plasmid pRDB_052 (for stable fly cell... pRDA_791 (contains only orio and ampR) Drosophilia Plasmid pRDB_051 (for stable fly cell lines...integration by spontaneous insertion)   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy...

...Table 1 has a detailed list of the bacterial species these plasmids are compatible with.  Table 1: Non-...reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https://doi.org/10.1016/0378-1119(88)90117... N.J.), 765, 327–343. https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and ...Gluconacetobacter xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus...Escherichia coli, Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens,... A. viscosus, Aerobacter aerogenes, Aeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type...

...of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using...highly cited, we went to one of the largest bibliographic databases available, Web of Science. For each... publication, Web of Science provides basic bibliographic information and uses its vast network of articles...

...BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based fluorescence...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...study model organisms such as Drosophila and zebrafish. Brainbow in Drosophila has aided in the mapping of...tracing in other systems, such as zebrafish and Drosophila. Why Brainbow? Brainbow highlights the trend ...

...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...RBPs successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This...first step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that ...in the bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to...proteins expressed by your library. After blocking for 1 h at room temperature with gentle shaking, gently ... to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding of the recombinant...

... 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,.... They can also be introduced directly into a Drosophila S2 cell line stably expressing Cas9. This PCR...Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells.” G3 (Bethesda) 6(6) (2016...

...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...microscopy: a brief history and new avenues. Philosophical Transactions Royal Soc 380:20210110. https:/...

...gene expression and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518...Expression System for Drosophila. Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central...implemented in a variety of other organisms, such as sphingomonads and bacillus.  ADH1 The ADH1 negative repressible...culture. Repressible Binary Systems GAL4/UAS In Drosophila or development studies, you may hear some transcription... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further...

...infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous recombination...sequences of interest to cells with efficiencies of 1-40% after G418 selection (Kohli et al 2004, Kim et...with polylinkers for the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor...since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there are ...efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%. While this is comparable...gene inactivation in human cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene...zygotes and thereby correct a mutation that causes Hemophilia B. Unfortunately, work combining CRISPR with ...

...incorporates several common genetic tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding...Rodríguez et al., 2011).  Genetics of SPARC Figure 1: SPARC and SPARC2 use the GAL4-UAS system along with.... So far, tools to overcome this challenge in Drosophila require heat-shocking the system or using chemical...techniques to reliably label any post-mitotic cells in Drosophila. SPARC was developed by Thomas Clandinin’s lab...recombination sites. These sites are target sequences of PhiC31. In the SPARC system, attP sites are located on...is not expressed and the cells are not labeled. PhiC31, a recombinase. It specifically integrates an attP...attP site into an attB site. In the SPARC system, PhiC31 irreversibly recombines one of the two competing...

...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; ...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors...is multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky... addiction. When an animal has been exposed to morphine or other stimulating drugs, it develops an enhanced... such genetic study focuses on brain derived neurotrophic factor (BDNF). BDNF is a small protein involved...