...light. Activation of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission... but now we’ll focus on how viral vectors have changed the scope and power of three commonly used behavioral... confirms the previously suggested inhibitory mechanism of locomotor sensitization and might open new ...the open (risky). Taking advantage of these characteristic traits, rodents can be used to study anxiety...mouse to deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that...Central PMCID: PMC6213283. Bahi, Amine, Vijay Chandrasekar, and Jean-Luc Dreyer. "Selective lentiviral-...

...base precisely? Until 2016, precise single base changes were only possible through exploiting the homology-directed...efficiently than before. A base editor precisely changes a single base with an efficiency typically ranging...ranging from 25-75%, while the success of precise change via HDR limited to 0-5%. This blog post covers ...wonder if a CRISPR-base editor can perform this change. However, there are no naturally known 'DNA adenosine...used to detect success. Although there is a slim chance of generating indels with base editors, keep in... 627-630. PubMed PMID: 29476916. 10. Kang, Beum-Chang, et al. "Precision genome engineering through adenine...plants (2018): 1. PubMed PMID: 29867128. 11. Li, Chao, et al. "Expanded base editing in rice and wheat...

...concept for further development. Figure 2: Mechanisms of deaminase-based adenine transversion editors...editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. Base ...Acronym Full name Notes   Base transition Base change between purines (A ↔ G) or between pyrimidines ...pyrimidines (C ↔ T).   Base transversion Base change from purine (A or G) to pyrimidine (C or T) or vice-versa...doi.org/10.1038/s41587-023-01821-9 He, Y., Zhou, X., Chang, C., Chen, G., Liu, W., Li, G., Fan, X., Sun, M....M., Miao, C., Huang, Q., Ma, Y., Yuan, F., & Chang, X. (2024). Protein language models-assisted optimization...Li, J., Ren, J., Zhang, X., Zhang, Y., Tang, H., Chang, X., Yu, Y., & Wei, W. (2024). Programmable DNA ...

...cell-type and temporal precision. In ICSS, a test chamber is used in which a nose-poke at a particular hole...but is considerably simpler than ICSS. The test chamber is divided into two halves that are distinguished...animal only receives stimulation in one of the two chambers (see image below). If a circuit is reinforcing...animal will prefer to spend most of its time in the chamber in which it receives the stimulation (conversely...it will spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...specific behavior but that they more precisely characterized with spatial (CA1), temporal (real-time inhibition... the field benefits from collaboration and the exchange of ideas. The rapid spread of optogenetics throughout...

... problem, how can one little change make a difference? Individual changes to our behavior can lead to ...By initiating little changes in ourselves, we can both influence others to change their behavior and we... Recycling and Innovations posts. Making small changes in your daily habits can feel ineffective or unimportant...Green Lab also has resources and even a freezer challenge that your university could join. Find alternatives...

...this rapidly changing environment. In particular, we are working to understand what challenges remain in ...Addgene saw big changes in 2022, from the launch of a new material to the redesign of a community resource...around the world, are learning to navigate the changes brought about by the pandemic. As we adjust, we...learn from, and a compelling future to plan in the changing landscape of science. We are therefore embarking...to serve the scientific community as we grow and change to meet its ever-evolving needs. For now, we have...

...domain is interesting because by changing certain amino acids you can change the specificity,” explained Zhanar...she hopes will change the intracellular localization of RNA. “You can think of changing bases to other... Abil of the University of Illinois at Urbana-Champaign. The PUF proteins include eight modular repeats...binds a consecutive base in its RNA target. By changing amino acids, it’s possible to produce a protein...

... #1, any change in the normalized intensity of your POI could reflect either a genuine change in your ...your POI, but simply whether its concentration changes in response to experimental parameters. This is... intensity. Researchers use later steps in this chain (blue) to infer or calculate things about earlier... even hit the gel. But what if your experiment changes the expression of your loading control without ...your POI or an opposite change in your loading control. Assumption #2: The loading control can be accurately...become extremely susceptible to error, since tiny changes in intensity can reflect massive differences in...

...see below). Importantly, although their role has changed, restriction enzymes are certainly not dead yet...Introduced in 1977, Sanger sequencing, also known as chain-terminating or dideoxy sequencing, was a major breakthrough...biology98.3 (1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new prenatal...hybridization for detection of gene copy number changes." Protocols for Nucleic Acid Analysis by Nonradioactive... PubMed Central PMCID: PMC2743315. 5. Roberts, Richard J. "How restriction enzymes became the workhorses...Nicklen, and Alan R. Coulson. "DNA sequencing with chain-terminating inhibitors." Proceedings of the National...: PMC431765. 8. Heather, James M., and Benjamin Chain. "The sequence of sequencers: The history of sequencing...

...Precision genome editing faces the challenges above and more. One chief challenge is upregulating HDR in mature...complex oligonucleotide treatment comes with many challenges, researchers have begun to explore genome editing...approaches for exon skipping. Addgene depositor Charles Gersbach used paired zinc finger nucleases to remove...highest-ranking predicted off-target sites. Each paper characterizes additional, unique facets of CRISPR DMD therapy... the time to clinical approval. Even with the challenges precision editing faces, it’s encouraging to ...Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya S, Shelton JM, Bassel-Duby R, Olson EN. Postnatal...

...Content Accessibility Guidelines (WCAG). These changes mean our content is both available and accessible...expanded a growing collection of thoroughly characterized antibodies, and our contributions to key research...building a portal to easily share sequences and characterization data for over 150,000 plasmids, empowering...closer to making an impact on humanity's greatest challenges. Thank you for being part of this incredible ... (2024). A consensus platform for antibody characterization. Nature Protocols. https://doi.org/10.1038...Cable, H., & Longworth, S. (2024). Antibody characterization is critical to enhance reproducibility in ...

...engineering, Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier. I was joined by Addgene Scientist Matt Ferenc...and Joan Steitz. This award ceremony honored Charpentier and Doudna, who talked about their research leading... leading up to the discovery of the mechanism of dual-RNA-guided DNA cleavage in adaptive bacterial immunity...simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at a 2011 conference and decided to collaborate... project started with their labs studying the mechanisms of RNAi, the field for which Craig Mello and ...was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the crucial role of basic research...

...researchers involved. Communication is a major challenge when researchers are communicating across disciplines...disciplines or doing different tasks. But these challenges can be exaggerated when communicating across ... help other teams successfully overcome these challenges, too. Schedule regular meetings Scheduling a...person Presenting the results from my lab exchange project at the International Cancer Immunotherapy...other in person. One possibility is to do a lab exchange, where researchers spent time in each other’s ...performed the same way at different sites. I did a lab exchange as part of the collaboration I led. This gave ...

...just how much one’s environment changes them. Addgene gave me the chance to become a real life scientist...wanted to major in biochemistry. There, I had exchanged contact with Amy C., Addgene’s intern coordinator... — such as using Google workspace — and got the chance to work on my very own research project! Coming...scientist. Addgene gave me the chance to grow into a community that had opened its arms to me from day one. .... Addgene has changed my life for the better, and I wouldn’t want it any other way. Toj Toj-Villagran ...

...variable domains of heavy chain only antibodies) (B) Nanomice express hybrid heavy-chain only antibodies that...’s spotlight after researchers discovered heavy-chain only antibodies in the serum of a camel and soon...)!      Figure 1: Comparison of a heavy-chain only antibody and single-domain antibody, under ...need for a species that naturally produces heavy-chain only antibodies. (A) Overview of genetic modifications... way since the serendipitous discovery of heavy-chain only antibodies in a camel. And I suspect we will...

... approaches a host cell surface are complex polysaccharide structures, many of which are easily varied... to be explored and exploited. With better characterization, these proteins could function as surrogate...surrogate antibodies for identifying and characterizing sugars and sugar-containing molecules in molecular biology...and pressure. Nebulization units, which can be purchased cheaply, are small plastic containers that can... can perform whole genome amplification (by a mechanism known as multiple displacement amplification) ...cloning site of the vector. In order to reduce the chances of some clones being outcompeted by others in liquid...colonies of interest, sequence the inserted DNA and characterize your new putative host cell-binding proteins...

...gel causes the negatively charged proteins to migrate toward the positive charge at the bottom of the gel...and the protein is separated through a matrix by charge. In a denaturing western, the protein is denatured...buffer. These systems, however, require you to purchase expensive system-specific sandwiches.   ...while a western blot detects denatured polypeptide chains. The protein regions available for antibody binding...protein in its native state versus the polypeptide chain. Consequently, antibodies validated for IP might...addition to protein abundance, westerns detect changes to a protein’s post-translational modifications...

... material, and variations between serotypes can change how the viral particle sneaks its way into cells...Citation CNS AAV1, AAV2, AAV5, AAV8, AAV9, AAVrg Aschauer et al., 2013 Heart AAV9 Bish et al., 2008 ...expertise! References and resources References Aschauer, D. F., Kreuz, S., & Rumpel, S. (2013). Analysis...., Yin, L., Merigan, W. H., Flannery, J. G., & Schaffer, D. V. (2013). In vivo-directed evolution of a...Y., Jae, L. T., Wosen, J. E., Nagamine, C. M., Chapman, M. S., & Carette, J. E. (2016). An essential receptor...Gaj, T., Lavzin, M., Ritola, K. D., Lindo, S., Michael, S., Kuleshova, E., Ojala, D., Huang, C.-C., Gerfen... Dudman, J. T., Hantman, A. W., Looger, L. L., Schaffer, D. V., & Karpova, A. Y. (2016). A Designer AAV...

...trees and comparing them with the gene expression changes of more than 100,000 cells undergoing iEP conversion...tags often had similar gene expression pattern changes and reprogramming outcomes, suggesting that outcomes...results suggest that iEP reprogramming is not stochastic, but rather there’s a particular cell state that...successful iEP reprogramming. MettI7a1 encodes an uncharacterized putative methyltransferase. Adding Mettl7a1... expression and clonal history associated with changes in cell identity. The Morris Lab used CellTagging... processes where cells are undergoing identity changes, such as the differentiation of induced pluripotent...

...Leiwe and Takeshi Imai from Kyushu University. Stochastic multicolor labeling is a powerful solution for...microscopy-based neuronal reconstruction. To achieve stochastic multicolor labeling, Brainbow used the Cre-loxP...copies of the transgene cassette are introduced, stochasticity will result in a combinatorial expression of...Brainbow was inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins... such vector-based gene delivery systems, the stochastic expression of XFPs is possible by simply introducing...introducing a mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes...

...However, these approaches are often met with the challenges of regulatory bottlenecks and their scope is ...is limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision... various metabolites in Escherichia coli or Saccharomyces cerevisiae with few techniques that are easily...designed scaffold binding sites. With hundreds of characterized aptamer domains available, an unprecedented .... This strategy can be applied to effectively change metabolic flux. The dCas9 protein and orthogonal...6441/ Resources: An open source library of characterized aptamers and supporting publication: http://...

...they change color as they shift from "off" to "on" in the presence of calcium. This color change can be...problem that he considers “one of the greatest challenges of neuroscience.”  Hot plasmids: Twitch sensors...imaging neurons, Griesbeck adds that this color change has a particular advantage for observing events...sensitivity. As reported in Nature Methods, the FRET changes of these Twitch sensor variants were optimized ...trial and error. Transition metals also present challenges in that they are found in cells at much lower...

...through filtration methods because of its ability to change shape and the absence of a rigid cell wall. Here.... For testing, the medium should not have been changed for at least two or three days before sampling ...chemical reactions and this induces a noticeable color change. This method has high sensitivity but low specificity...specific enzymes are used which trigger cellular mechanisms that generate luminescence. This can then be ...are working without gloves or masks. Make sure to change the gloves and masks regularly instead of keeping...water provided to maintain the humidity should be changed regularly as mycoplasma, bacteria, and fungus can...Mycoplasma Contamination in Cell Cultures by Polymerase Chain Reaction. In: Methods in Molecular Biology. Humana...

...questions in the chat box. That’s another benefit to note-taking: you can revisit the Q&A in the chat box. Presenting... plan more with regard to my interests.” Use the chat box and upvote If the thought of walking up to the...asking questions using the polling feature or the chat box. One key thing to remember regardless of the...poster,” he says.Viewing a poster on a screen is challenging, requiring you to zoom in to different parts ...than in-person. You might miss out on the short chats between sessions or waiting for a session to start...

...years. They helped one another navigate career changes and overcome barriers by sharing broader perspectives...Healthcare Businesswomen’s Association (HBA) Boston Chapter and the Massachusetts Association for Women in ...diversity of input and perspectives Provides a good mechanism for accountability that makes mentoring effective... heard from peer groups meeting at both the Fox Chase Cancer Center and Brandeis University. 5 easy steps...choosing a topic and leading the discussion. This is a chance to practice leadership and communication skills...better with process, so don’t just get together and chat. Have an agenda and get everyone ready for the discussion...

... simultaneously   Researchers in the labs of Michael Lin and Ryohei Yasuda have teamed up to develop...maturation. Three new RFPs were isolated and characterized: mScarlet (fluorescent lifetime of 3.9 ns, quantum...member of this class that works via a different mechanism - the PhoCl protein is irreversibly cleaved when... also provided a detailed protocol at Protocol Exchange. Plasmid maps can be found at Addgene and in Figure...Gram-positive bacterium, many of its genes are poorly characterized. The Gross lab hopes these libraries will be...Transcription factor fusions elucidate iPSC reprogramming mechanism The Ptashne lab was the first to demonstrate ...used this now classic technique to elucidate the mechanism by which SOX2 acts to reprogram human fibroblasts...

...Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed ...representing a protein super family). To overcome these challenges, we set out to develop a hassle-free, highly ...Technologies, USA). These synthetic RNAs can also be purchased from various other suppliers with a typical turnaround...straightforward as the RNA molecules can be directly purchased and there is no need to construct plasmid vectors...; this was possible because reagents could be purchased directly from commercial suppliers without investing...template) and use homology arms of 60 -70 bp . Purchase gene-specific crRNAs and matched ssODNs from commercial...by single-cell deposition in 96-well plates: Characterize clonal lines by ICC and PCR genotyping (Sanger...

...Many thanks to our guest blogger, Doug Richardson! Doug Richardson is the Director of the Harvard Center...This post was contributed by Doug Richardson, Director of the Harvard Center for Biological Imaging and...microscope designs may further exploit the unique characteristics of fluorophores to block out of focus fluorescence...blurry out-of-focus light. The most difficult characteristic to capture in these types of samples is their...model embryos >15 μm Thsee are some of the most challenging samples to image as they require both optical... 2, 932-940 (2005). PubMed PMID: 16299478. 9. Richardson DS, Lichtman JW. Clarifying Tissue Clearing. ...

...and RNA expression - but what if you just want to change protein levels? Until recently, no such tool existed...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...the first author of this article, Dr. Douglas Richardson, recently contributed a relevant post to the ...Addgene blog. Find the SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID:...transcription factors in series and independently changes inducer levels. That is, if transcription factor...expression of a reporter protein, it is possible to change the amount of noise in reporter gene expression...

...lack of caveolin 1 in breast cancer also induces changes of stromal cells in the microenvironment. This ...from cancer cells to the microenvironment. This mechanism happens for both short and long distances. When...transferred to other cells, inducing epigenetic changes in targeted cells. In this case, exosomes may transport...extracellular matrix and fibroblasts. With each cell mechanism discovered in microenvironments, we are one step...References Allinen, Minna, et al. "Molecular characterization of the tumor microenvironment in breast cancer...Intercellular nanovesicle‐mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular ...

...•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes, especially in...hemoglobin. Previous studies have shown that one base change in the promoter region of gamma hemoglobin (HGB1...effects. ABEs have been described to introduce base changes in cellular DNA and RNA dependent or independent...authors tested ABE8.17-m and found that one base change mutation (ABE8.17-m+V106W) (Gaudelli et al., 2020...

...in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined the function ... Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease...CRISPR-Based Approaches for the High-Throughput Characterization of Long Non-Coding RNAs. Non-Coding RNA. 2021.... doi:10.3390/ncrna7040079 Horlbeck MA, Liu SJ, Chang HY, Lim DA, Weissman JS. Fitness effects of CRISPR...(5):573. doi:10.1038/s41587-020-0428-0 Rinn JL, Chang HY. Genome Regulation by Long Noncoding RNAs. Annual...in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front Plant Sci. 2015;6. ...

... heteroduplex is cradled in the positively charged channel between the two lobes. For CRISPR to be programmable...light blue; bridge helix, purple.   Apo Cas9 changes conformation frequently (Osuka et al., 2018), but...structure, reflecting the less stringent recognition mechanism that can tolerate mismatches in this region. The...in the duplex that may not fit properly in this channel, depending on their precise location. The end of... BioRender.com.   The exact conformational changes that make Cas9 such a valuable laboratory tool ...Doudna, J. A. (2017). CRISPR-Cas9 Structures and Mechanisms. Annual Review of Biophysics, 46, 505–529. https...

...institutional oversight is required. Restraint: mechanical restraint or anesthesia Location: tail vein or...you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume...xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind legs Gauge: ...Restraint Location Gauge Volume Skill Intravenous Mechanical restrainer or anesthesia Tail vein or retro-orbital...cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer Thigh 25–27 < 0.02–0.05 mL Beginner...Beginner Subcutaneous Back scruffing or mechanical restrainer Back or hind legs; between skin and muscle...

...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material is damaged...daily, yet obstacles remain. One of the biggest challenges facing researchers today is the complexity of... of the latest resources? In response to this challenge, Addgene has launched an initiative to curate ... your thoughts in the comments section below. Michael G. Lemieux is a Scientist at Addgene with interests... Michael G. Lemieux...

...Did you notice the “Filter By…” options changed when we changed from searching for viral preps instead ...best plasmid for your research can be a daunting challenge. With over 80,000 deposited plasmids from around...assembled a series of screenshots to highlight changes to both the design and functionality of searches...of plasmids? The available filtering options now change dynamically based on the type of material and specific...you may find the available categories and filters change as we now only show options relevant to your current...

...Disable the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous...mammalian cells, but a less frequent DNA repair mechanism in general. On average, it accounts for less than...is a silent mutation, so you don’t accidentally change your protein of interest.   Fig. 2: Stop...potentially shuttling repair down a different pathway (Charpentier et al., Nat Com).     Fig. 3: HDR events... 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion, A...

...again and possibly mediate HDR. This “second chance” mechanism might improve the frequency of desired HDR...al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage...requirement. Zetsche et al. started from square one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (...elements of CRISPR-Cpf1, Zetsche et al. turned to characterizing its cleavage pattern. Again, they were in for...efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs...

...research. This includes chat apps like Slack, Microsoft Teams, and Google Hangouts Chat, resources like Github...an electronic environment and hence making it searchable and shareable, but some ELNs provide better support... delve deeper and look specifically at the characteristics and capabilities an ELN needs to facilitate...send documents from RSpace to a selected Slack channel for discussion and review. It’s also possible to...many different ELNs out there can help with searchability, record-keeping, and data sharing, not all of...

... LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL...reporter is added to the reaction. Cleavage of the quenchable fluorescent RNA by the “activated” Cas13 produces...Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti... Koonin EV (2015) Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular...