...HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central PMCID: PMC6610143...fused to a fluorescent protein. “Frankenbody,” like the infamous frankenstein, was constructed by grafting...called this probe the HA frankenbody (Zhao et al., 2019).  To develop HA frankenbody, the Stasevich lab tested...can you do with the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound...also proved that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA...acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ability to...properly once inside cells.  Developing the HA frankenbody To combat the issues with these antibody based...

...tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope... 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al., 2005)     References and ... on different viral membranes, tropism, and advantages and disadvantages: Table 1 (Joglekar et al., 2017... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...virus to either restrict or broaden the host cell range (tropism). Pseudotyping is only done on enveloped...plasmid, and (3) the packaging plasmid. These are transfected into target cells which produce lentiviral particles...

...holds true in genome-wide screens.   Figure 1: Summary of the advantages of the new approach to ... in both screens, with the growth screen having a 1% false discovery rate and identifying >88% of previously...in Morgens et al. contains three types of guides: 1) targeting guides (blue), which target all protein-coding...off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets had greater toxicity than ...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated than.... When comparing the toxicity of guides that have 1-bp mismatch off-target sites, short guides had reduced...Knockout Library are available from Addgene! References 1. Morgens, David W., et al. "Genome-scale measurement...

...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...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ...

...). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...serotypes 1, 8 and 9 share conserved mechanisms for anterograde and retrograde axonal transport. Hum Gene.... (2009). Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat ... Brain Res 406(1-2), 317-321. PubMed PMID: 2436717. Towne, C., Schneider, B.L., Kieran, D., Redmond, D.E.Jr...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...

...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in ...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ...

...842 . https://doi.org/10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, ...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous...

...the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below) ...complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA duplex. Image from...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...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...

...vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles ... are often found with cellular exosomes, small membrane vesicles or microvesicles (approx. 40 - 150 nm... diameter) that are secreted from the cellular membrane into the culture medium. AAV particles are either... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...to modify it to improve yields. For example, transfecting the producer cell line with an expression vector...proteins, nucleic acids) which possibly improve transduction properties. György and colleagues found that...that the association of AAV particles with membrane vesicles protects them from antibody recognition and...

...methods to improve AAV production.     References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high...be required. For example, retrograde AAV gets transported up the cell’s axon. While the mechanism for this...yet fully understood, we can imagine that this transport is probably not 100% efficient. So the few AAV... higher retrograde AAV titers for successful transduction. In another example, neuronal gene expression..., fewer channel proteins are required for the transduced cell to become light responsive than fluorescent...process of generating viral particles. Limit tube transfer - the fewer opportunities your viral particles...

...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little as 30...Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.  Additional Resources on the Addgene...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...

... microscopy contrast technique in modern biology (1). When performed correctly, fluorescence microscopy...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...

...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...material is right for their experiment.     Table 1: Most popular guides, protocols, and collections of...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... package stays in transit for a certain number of days and tracking average transit time for each courier...

...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...this type of experimentation to the eventual translation to the clinic and cures. There is no doubt that...

...Issue 19, 1 October 2000, Pages 3752–3761, https://doi.org/10.1093/nar/28.19.3752Wong M; Medrano J. Real-time...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...in primer Yes Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from...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...

...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 ...Gate cloning, perform an exonuclease cleanup, transform, plate, and pick. In short, the Fragmid system...AAV pRDA_889 (ITRs from AAV2) Mammalian Transposon pRDA_757 (Piggybac; constitutive expression...   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy insulator sequences; white + selectable...

...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...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...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition...

...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...proteins, which could disrupt your target epitope (Fig. 1). So, while organic solvents may improve antibody ...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created...

...used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey The take-...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the survey. When asked... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...Doudna Lab Many thanks to our Guest Blogger Pooran Dewari! Pooran Dewari is a postdoc at the MRC Centre for...

...). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology,...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... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton...

...pitfalls? Here's what you need to know.   Figure 1: Management can seem a mystery at first! Image used...Understanding the transition While a PhD equips you with exceptional technical skills, transitioning into management...   Strategies for a successful transition To successfully transition into a managerial role, it's essential... most pivotal decisions many PhDs face when transitioning from academia to industry is whether to stay...career trajectories within the industry, but the transition wasn’t easy - I had to reinvent myself and redefine...d like to share below. Common pitfalls in the transition (what I wish I knew before experimenting on myself...being a great scientist doesn’t automatically translate to being an effective manager. Mismanaging (no...

...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...up to 10-12 fragments (Pryor et al., 2020), and randomly selected non-palindromic pairs allows for high-fidelity...

...premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells Complexes...  HeLa, NIH3T3, CHO, COS-1, 293HEK Inhibits termination step of translation 2-10 ug/mL G418/Geneticin...expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T ... does not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...

...here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around...start out at our Cambridge office – previously at 1 Kendall Square, but newly relocated to 75 Sidney Street...Have a question? Email: [email protected] Call: +1 (617) 225-9000         ...Addgene's lab. We ship individual plasmids as transformed bacteria in stab culture format. Our lab team... and collaborations with institutions and tech transfer offices that facilitate MTAs. The great feedback...

... advanced and finally, to expert.   Figure 1. Visual representation of common injection routes ...back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner Intradermal ...work. Good luck with your in vivo experiments! Table 1: Summary of common injection routes in mice. Injection...hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between...resources on the Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms... 0.07 ml Skill required: advanced Intranasal instillation Intranasal instillation is very similar to its...behind the eye. IV injections are used for a broad range of substances, as it circulate what is injected ...

...system defined three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open...deletion of release factor 1, the protein responsible for terminating translation in response to the presence...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,...

... successful and meaningful career in the grants profession: 1. Build on your existing skills & passion...Associates, Inc., a national grant consulting firm, and Smartegrants, a grant professional development firm... the field of grants. What is a grant professional?  In the past 5-10 years, the Grants Profession has...readiness, identify grant opportunities, secure grant awards, and manage grant projects and funds. You will... for grants, the amount of grant funds available and the increasing regulatory process for grants has ...become a grant professional? Employment opportunities for those with expertise in grant writing, grant management...as a Grant Professional to mentor and coach you. There are many grant consultants who offer grant training...

...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...://www.linkedin.com/in/kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of...

...the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It is a Drosophila geneticist...still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression as a...Elliott, A. H. Brand, The GAL4 System : A Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus...Lai, T. Lee, Genetic mosaic with dual binary transcriptional systems in Drosophila., Nat. Neurosci. 9, 703...

... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break by homologous recombination...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...repair of DNA double strand breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372...(single vs. double stranded) all depends on the size of the knock-in. Single strand oligonucleotide donors...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed...

...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources..., and deletions) without introducing DNA double-strand breaks (Anzalone et al., 2019; Chen & Liu, 2023...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...

...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...Image reproduced from Frank Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie ...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...tool that responds strongly to magnetic fields (Frank Hayward et al., 2024). They deposited their plasmid...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist...when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence intensity...

...simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special...allow immediate co-transformation with linearized vector backbone. DpnI digestion - 1 μl DpnI is added...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...

... class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was ...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided... Cas9’s shortcomings - namely its blunt double stranded cleavage and G-rich PAM requirement. Zetsche et...one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid...

...Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought...Cre 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...project to the NAc, where enhanced glutamate transmission leads to locomotion sensitization. (for review... Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC projections to the NAc and decreased...

...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...(s) Cell lines Cell lines, primary cells Table 1: Key differences between original CAPTURE and CAPTURE...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu...sequences that, when bound by transcription factors, can increase a gene’s transcription. Sometimes enhancers ...and proteins that forms chromosomes. These long range DNA interactions are often detected with chromosome...

...that have now become hallmarks of ideal FP timers: 1. Ratiometric determination of expression time The ...the FT series matures from blue to red (see Table 1 for emission and absorption spectra). Subach et al...times and varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring...cellular events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from ... know in the comments section below! References 1. Terskikh, Alexey, et al. ""Fluorescent timer": protein...production of dsRed E5. This timer predictably transitions from green to red fluorescence (500 to 580 nm... of vesicle fusion and release from the plasma membrane by fusing various vesicle cargoes to FP timer ...

...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)...epithelial origin. Suppressing function, creating tolerance, and evading recognition are a few methods by ...

...) element and the cis-acting EBNA-1 from Epstein-Barr virus. EBNA-1 binds oriP and allows for replication... plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming...reprogramming proteins. Table 1: Key Features of Different Methods for Generating iPSCs. One important...  Replicating oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These plasmids carry the origin...method Most iPSC research falls into two categories: 1) studies focused on better understanding the mechanisms...iPSCs experiences in the comments below! References  1. Sano, Masayuki, Minoru Iijima, Manami Ohtaka, and...Methods figures were adapted from this reference. 1. Malik, Nasir, and Mahendra S. Rao. "A Review of the...

...much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR...one NHEJ-repaired indel, often occur. Figure 1: In conventional gene targeting (A), you must first... drug induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid...CRISPR ribonucleoproteins.” Genome Biol. 18 (2017) (1):92. DOI: 10.1186/s13059-017-1220-4 PubMed PMID: 28511701... using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038/nprot.2017.153 PubMed PMID:...engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059-018-1400-x PubMed PMID: 29482575... Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have been shown...

...from the paper for designing experiments with AAV: 1. Getting your viral vector into your cell of interest... administration routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest...information from a region. In the example shown in Figure 1, injected retrograde AAVs at one location result in... cell types. Specific transgene expression can be also achieved in nontransgenic animals by using cis ...delivery by increasing transgene cargo capacity, enhancing anterograde transport efficiency, and engineering...proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to the target...serotype used for a specific cell type. Find some transduction characteristics of select AAV serotypes in Table...

... Effectors.” Cell (2018) pii: S0092-8674(18)30207-1. PubMed PMID: 29551272 Plasmids from this paper ...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...

...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ...(p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose) without signs of ...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ...the transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent... These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293-TETOn...

...strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes deleted. ...that the culprit for these unusual cases was either 1) a physiological effect of SARS-CoV-2 toward destabilizing...certain contexts (Bauzon et al., 2003).        Figure 1: The adenovirus life cycle. Image reused from Kremer... review of the evidence. Emerg Microbes Infect, 8(1), 1679–1687. https://doi.org/10.1080/22221751.2019.1690953...574–580. https://doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval...host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped double-stranded DNA viruses... protein translation. Its genome consists of “early” (E) and “late” genes, and the transition between ...

.... Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into...: promoter, gene, and terminator.     Fig 1. MoClo enables individual parts to be assembled into... peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...into… Transcription units (TUs), which are functional units composed of multiple parts transcribed together...a protein that regulated its own transcription, or the transcription of another gene? Now you have a genetic...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you...hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs. Let’s start...

... The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There are eleven naturally...closer look at the rep and cap genes. Figure 1. The RepCap plasmid and its products. Created with...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...and cap are expressed from a separate plasmid in trans, freeing up space in the viral genome so that a ...Replication initiates from the AAV genome ITRs on the transfer plasmid, which contain regions of dsDNA. To generate...capsid. Rep proteins are also involved in the transcriptional regulation of the AAV p40 promoter which drives...promoter. Alternative splicing and differing translational start sites generate the three VP proteins. ...

...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y,...harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs, in collaboration...from the University of Pennsylvania.     References: 1. Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions...details how to transfect CRISPR plasmids into MEL cells by electroporation, sort your transfected cells via...CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety of loci. We were...following paired DSBs is inversions [3]. These rearranged segments may also be informative as to genomic...

...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...potential hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken...where the topic of your research can make your transition easier – choose a lab that works on problems ...

...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...

... blocking translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result...dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target... III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and...in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...