...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site will...genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O...switches? FLEx (or “flip-excision”) switches were designed as a genetic tool for researchers to conditionally...does the FLEx switch work? Let’s say you want to design a genetic FLEx switch that turns BFP expression...

...is right for their experiment.     Table 1: Most popular guides, protocols, and collections of 2023. ...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico...in 2023, from visitors in 218 countries. In Table 1, you can see our most popular educational resources... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid...shipping. However, there were issues that persisted despite our best efforts. With a little over half of our...

...Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors... Let's walk through designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector...answers in step 1, so you’ll only be able to select components that work with your design. Note that you...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden...website, which breaks the design process down into four easy steps, provides the real value for the user...potential of them not working, or spend a few weeks designing and cloning the vectors you actually want. But... Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach to cloning...

...materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created ...EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel...Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...case, a buffer exchange can be performed using a desalting column or an ultrafiltration concentrator. As ...suggests, you may also concentrate your prep to the desired concentration using these columns. If you are conjugating...

...For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one expressing Cpf1 and the other...comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1 CRISPR Nucleases   ...Overall, these approaches have two main drawbacks: 1) Most rely on transfection of more than one vector...leave them in the comments below.        References 1. Zetsche et al. "Multiplex Gene Editing by CRISPR–...editing!  A recent paper from Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  ...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing....  Similar to a jigsaw puzzle, the oligos were designed with sticky ends that only anneal together in ...

...not let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists...take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone with no interesting discussion...introductions. The moderator asks questions, calling on 1-2 panelist for each question in a way that elicits... time you are at a talk and the speaker goes over 1 hour, watch what happens to the attention of the audience...postures shift, they are checking out. If you can, plant 1-2 good questions with trusted audience members. A ... asking the panelists not to use any slides for the event. Slides are for presentations, not discussions...discussions. I will rarely work with a panel if slides are allowed. Don't put your panelists behind a table. ...

... in modern biology (1). When performed correctly, fluorescence microscopy provides a high contrast image...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...of modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...

...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...Wolfe, J.H. (2014). Adeno-associated virus serotypes 1, 8 and 9 share conserved mechanisms for anterograde...,9 in rat neurons and glia in vitro. Virology 372(1), 24–34. PubMed PMID: 18035387. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...

...it, select your desired serotype and volume, and submit your request.    Figure 1: The AAV Packaged..., AAV8, or AAV Retrograde (AAVrg), and volumes of 1 mL, 2.5 mL, or 5 mL. Please note that all orders will... our on-demand viral service, we expect titers of 1 × 1013 GC/mL for AAV1, AAV5, AAV8, and AAVrg and 7...simply request your plasmid of interest in your desired serotype, place your order when ready, and we’ll...

...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)...can be used for specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because...pseudotyping allows these viruses to infect neurons. Besides altering the host tropism of your virus, there ...neurons. However, VSV-G’s broad tropism can be undesired for in vivo studies since there’s the potential...VSV-G is also toxic at high concentrations as described above and is inactivated by the complement system...

...been designed to perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an ...use is only likely to grow in the coming decades.  References 1. Kowarz E, et al. Optimized Sleeping Beauty...also important for social and regulatory reasons. 1.) The transposon consists of a mirrored set of inverted...T cells to target CD19. Immunol Rev. 2014 Jan;257(1):181-90. doi: 10.1111/imr.12137. PubMed. Find the ...

... Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design of primers...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak, indicating...changes in ΔΔCT are represented by RQs between 0 and 1. Flipping your experimental and control values during...see this fold change as a positive difference (RQ>1), which may be easier to conceptualize. Just remember... in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_3, © Springer Science+Business Media New... application, Bioinformatics, Volume 27, Issue 7, 1 April 2011, Pages 1019–1020, https://doi.org/10.1093...

... Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9 binding...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...who worked on this publication:         References 1. Richardson, Christopher D., et al. "Enhancing homology-directed...DNA post-cut. Using substrate DNA labeled on both sides of the break, we determined that Cas9 held tightly... single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and...approaches. Moreover, the cost of reagents is relatively modest, requiring expressed Cas9 protein, transcribed ... Blog Brush Up on Your HDR Basics Learn How to Design Your gRNA Find the Ideal CRISPR Software for Your...

...type of assay is a clonal cell line known to contain 1 integrated copy of the qPCR target gene. Developing... successful transduction experiment. References 1. Kutner, Robert H., Xian-Yang Zhang, and Jakob Reiser...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009... titration methods."BMC biotechnology 6.1 (2006): 1. PubMed PMID: 16836756. PubMed Central PMCID: PMC1534021...events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957. PubMed Central PMCID: PMC514534...virus will not only ensure that your infection is designed in the best possible way, but it may also save...the transgene itself. Many researchers prefer to design primers that target the common features of viral...

...Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed PMID: 26702451. PubMed Central PMCID...genetic manipulation in mammalian cells until 2005 [1]. Like other transposons, piggyBac™ has two components...The predicted frequency of ‘TTAA’ in the genome is 1 in every 256 base-pairs of DNA sequence, making it...has been demonstrated) with no known upper limit [1]. The piggyBac™ technology can be used for numerous...genetic loci of integration [8].   Figure 1: Mechanism of the piggyBac™  transposase/transposon...technologies and strategies for their projects. References 1. L. E. Woodard and M. H. Wilson, “piggyBac-ing models...facilitate the selection of cells containing your desired edit. The selection is marker is then seamlessly...

...minutes, which was fun to watch. A simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at...to direct cleavage the result was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the...with genes knocked out in the insulin signaling cascades in only 4 months compared to a year or greater...

...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...human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor ...

...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...introduce a desired genetic alteration. Listen to Our Podcast Interview with Alex Chavez Figure 1: Schematic...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The gRNA conventionally... truncated such that it exhibits 15 or fewer nucleotides of complementarity to a target site, Cas9’s DNA...truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated to result in decreased...

...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...