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... SPH mice CRISPRa has largely been limited to in vitro settings due to the large size of Cas9 activators...activators, which largely precludes delivery by adeno-associated vectors (AAV). This issue was recently circumvented...delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach was...neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church Lab. Neuron-specific...microglia are a notable exception) necessitates such molecular gymnastics for sensitive applications such as ...screening. The plasmids used by the Qi Lab for this particular application can be found here.  Why CRISPRa? ...well-suited, including studying mRNA dosing effects, cellular reprogramming, and disease modeling. Further, ...

...model organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ...In this heyday of molecular biology, many scientists do a lot of DNA work but never get to actually manipulate...materials sharing in their dissemination plans and in particular, that they all share the plasmids created under...which have been used to treat heart disease. Caterpillars that eat milkweed generate cardenolides that... poisonous) for bird predators - birds eat a caterpillar once, vomit, and never do it again. Cichlids ...Crepidula atrasolea juvenile, light microscope with polarizing filter. Development is direct in this species... fascinating social behavior. For example, two similar species have very different social behavior. One...

...important regulator of embryonic development The largest conference room at Addgene is named Sonic Hedgehog...segmentation during embryogenesis. Typically, regularly spaced denticles (bristles) outline the segments...contain SHH, you can find them all here! The most popular SHH plasmid however was deposited by none other...transposons as a non-viral system to integrate particular genes into mammalian genomes and easily disrupt...Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene called "...the fly actomyosin cytoskeleton that regulates cellular contraction. This network is composed of actin...find the exact origin of this gene name but the larval gut of bagpipe mutants vaguely resemble a bagpipe...

...understanding of the molecular behaviors of chemicals and proteins to learn how a particular drug or medical...novel drugs. Commercial Affairs teams make up a large and diverse group of professionals running the marketing...healthcare consulting firms can be involved in many similar activities depending on the expertise of the senior...challenging. For instance, the client might need to get a large amount of market research done to inform a go/no-go...might outline the impacts of combining targeted molecular therapies for cancer with chemotherapies, or developing... science and the commercial applications of a particular therapy. With your scientific knowledge behind... to a community of colleagues who are pursuing similar jobs and non-academic careers. Now, more than ever...

...datasets are too large to transfer to a journal repository, they are likely too large to be retrieved ... was necessary and reasonable. PLOS has since clarified their data sharing policy and recently announced...manuscripts that had been processed since the declaration, only a small fraction (<1%) of authors have ...their own data, with some smaller labs feeling that larger, better resourced, groups would harvest and reanalyze...efficiently trawled their own data? A distinct (perhaps similarly impoverished lab) might apply different or novel... PLOS’s credit, the organization subsequently clarified what the policy intent was and provided an FAQ...PLOS’ Bold Data Policy - by David Crotty (The Scholarly Kitchen) Read more Addgene blog posts about scientific...

...requested! Fire Lab C. Elegans Kit - Our first popular "kit," Andrew Fire's set of vectors for C. elegans... 2005. shRNA - In 2006, we saw an increase in popularity of empty backbones for shRNA expression, such...plasmids from Didier Trono's lab are our two most popular plasmids of the past 10 years. Induced Pluripotent...the most frequently requested. Ubiquitin - One particular gene we saw increased interest in from 2008 to...plasmid. A variety of plasmids continued to be popular years after they were added to the repository, ...activator-like effector nucleases (TALENs). Our most popular kit of the year was the Golden Gate TALEN Kit, ...Plasmids for fluorescent tagging have long been popular with scientists, but 2012 was marked by increased...

...birds spots the “caterpillar” and then poke out the snail’s eyes and ingests the larvae. The adult worms...biosynthesis in Drosophila melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed...snail’s eyestalks. Once ingested by the snail, the larvae reach the eyestalk and transform them into a pulsating...pulsating display imitating a moving caterpillar. The worm controls the snail’s behavior, forcing it out...parasite The parasitic worm Spinochordodes tellinii‘s larvae makes its home inside grasshoppers and crickets...

...should be widely applicable to other targets. The modular system of linkers and spacers could enable many...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...previously-reported technique for precisely integrating large DNA sequences into a genome (Anzalone et al., 2022...deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nature Biotechnology... (2024). Efficient site-specific integration of large genes in mammalian cells via continuously evolved...cells you are studying. Antibodies can help reveal cellular identities by targeting marker proteins that are...expressed in a given cell type. For example, glial fibrillary acidic protein (GFAP) is a glia-specific intermediate...

...sensitivity in the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For...fastest test that has been developed so far.  A similar method has been optimized to detect the SARS-CoV...molecule per milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C ...and will not give a positive result for highly similar viruses. Unlike Cas12a, Cas13a does not require...illuminator or UV light illuminator. Approach: Highly similar to the SHERLOCK diagnostic test, this test varies...identification using FnCas9 (FELUDA) Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment...Shravanti Suresh is a PhD candidate who investigates cellular aspects of CRISPR/Cas immune systems in the Sashital...

...Goedart, an assistant professor at the Section of Molecular Cytology and van Leeuwenhoek Centre for Advanced...Microscopy (University of Amsterdam). GFP is the most popular, most widely used genetically encoded fluorescent...fluorescent probe. Several factors contribute to the popularity of GFP including (i) fast and complete maturation...folded beta-barrel structure, followed by an intramolecular reaction that generates the chromophore (Tsien...Tsien, 1998). Importantly, this reaction requires molecular oxygen and as a consequence GFP remains non-fluorescent...Goedhart is an assistant professor at the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced...Kurt. "Genetically encoded fluorescent tags." Molecular biology of the cell 28.7 (2017): 848-857. PubMed...

... what your desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base ... applicable to knock-ins both large and small is a software very similar to TIDE known as TIDER (Tracking...deletion that isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels...edit. Most dual guide systems generate a deletion large enough that it can be visualized by running your...guide system.   Fig. 2: Size screening for large deletion events.   Validating knock-in mutations... Pro tip: make sure your PCR amplicon isn’t too large so that you can easily visualize the size shift ...

...dLight1, a molecular tool to detect and image DA in live animals (Patriarchi et al., 2018). Similar to calcium...the brain has been in vivo microdialysis of extracellular fluid (Darvesh et al., 2011). This technique... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...sensor, dLight1.1 and dLight1.2, exhibit high (submicromolar) DA affinity and high-magnitude responses (increases...s lab has created the GRABDA sensor based on a similar strategy: a GFP variant bound to a DA receptor,...cyclase in caudate nucleus of rat brain, and its similarity to the “dopamine receptor”." Proceedings of the...

...and targeting power of CRISPR combined with the large sequence capacity of transposons open whole new ...’t cause double-stranded breaks and can deliver large sequences and even entire genes. Figure 1...template and transposon proteins (Tns) to integrate large sequences into genomic DNA downstream of gRNA-specified...Created with BioRender.com. CASTs quickly became popular bacterial genome engineering tools, with early ... al., 2019). Type I-F CASTs were found to be particularly useful. But they were barely effective at editing...., Gelsinger, D. R., Vo, P. L. H., Chen, A. T., Tolar, J., Osborn, M. J., Sternberg, S. H., & Liu, D. ...on Addgene.org Addgene's CRISPR Guide (Jump to Large Edits) CAST Plasmid Collection ...

...encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined...Charles Gersbach’s lab at Duke University developed similar systems based on the light-inducible heterodimerizing...effector (LACE) system, the Gersbach lab utilized a similar strategy to develop an optimized photoactivatable...system was able to recognize the same PAM and had similar targeting specificity as full-length Cas9 (flCas9...light on and off. As one might expect from the modular nature of Cas9, Nihongaki et al. showed that it... respectively. The use of far-red light is of particular interest since blue light has limited applications...that post-UV exposure it showed cleavage activity similar to the wild-type Cas9. This photocaging technique...

...293, the most commonly used line expresses SV40 large T antigen and is thus dubbed HEK-293T. Given that...older cell lines in the research community, many cellular research tools have been optimized specifically... viral production because they express the SV40 large T-antigen. This allows them to replicate viral plasmids...HEKs can also grow in suspension, which allows for large scalability, and can also be adapted for serum-free...two areas of research where HEK cells may be particularly useful due to their intrinsic properties. ...by mycoplasma and bacteria, but HEKs are also particularly vulnerable to viral infection. Yes, your cells...Additional resources on the Addgene blog Five Popular Model Organisms 10 Tips for Mammalian Cell Culture...

...sometimes localize to unintended sites with sequence similarity to the on-target site. When a CRISPR-Cas system...frequency of 5%. Your experiment involves making a large sgRNA library to thousands of genes and targeting...targets by selecting a gRNA with low sequence similarity elsewhere in the genome. There are many predictive...Once you’ve selected a gRNA with low sequence similarity elsewhere in the genome to reduce off-targets...to consider your Cas enzyme. Even if there are similar sequences present in the genome, certain Cas enzymes...Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced. These sites can...appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would also identify all DNA binding...

...this latest work, the team designed and screened a large library of iGluSnFR3 mutants, identifying the two...somatosensory cortex, while iGluSnFR4s produces larger signal-to-noise ratios in two-photon imaging, making... making it ideal for video recordings of large populations of synapses and deep-brain fiber photometry...most experiments. These new tools promise to be a popular choice for a variety of synaptic imaging applications...gene makes stable cell line creation a breeze. Modular by design: Key elements (promoter, tag, polyA site...edits — single-base changes, small deletions, and large insertions up to 12 kb — with efficiencies between...isolating their associated protein interactome. Molecular Cell, 84(24), 4929-4944.e8. https://doi.org/10.1016...

...topics in biology, especially those at the molecular and cellular level. The reason for this is cost and ...it wasn’t until I started working in my first molecular biology lab in college that I really fell in love...limited budget. While there are more and more molecular biology-based activities making their way into... about operating the rest of the car as well. Similarly, cell free expression systems contain all the ...References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular synthetic biology education kit." Science Advances... to learn simple lab techniques Check out our molecular biology reference pages Find plasmids from the...

... by Alycia Bittner CRISPR pooled libraries are popular tools for genetic screens, enabling genome-wide...genome-wide interrogation of the genes required for cellular processes. For a successful screen, it is necessary...Schwiesow A leading cause of death worldwide, cardiovascular disease is driven by atherogenic lipoproteins...lipoproteins that accumulate within the vascular wall. ApoB, the main structural component of atherogenic lipoproteins...been shown to have a strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs...to aid in the understanding and treatment of cardiovascular disease. Find the LipoGlo plasmids at Addgene...78% in body wall muscle cells. These changes were larger than previously reported for other organisms, and...

...used to confirm the presence of an insert in a particular vector by excising the insert from the backbone...insert. You can search NCBI for YGOI to find the particular reference sequence if necessary. The example ...BamHI) are capable of cleaving sequences which are similar, but not identical, to their defined recognition... see 3 bands. This is due to the fact that the circular DNA takes on several conformations the most abundant... the DNA mass of comparably intense samples of similar size. Run the gel at 80-150V until you have good...the gel for longer to achieve good separation of larger DNA fragments. I hope these tips demonstrate ...More Plasmid Eductional Resources: Addgene's Molecular Biology Reference Find More Protocols and Videos...