... due to the interaction with quizzes and discussion on that week’s topic by the moderators. The assignments...scientists. Over the course of the class, the moderator discussed individual sections of a paper, the ...laborious, but helped build my understanding of how to generate a manuscript. At the end of the online course,...understanding of how to apply it. The personal interaction of critiquing others’ manuscripts as well as ...the writer works.  4. Practice writing everyday Overall, the most important instruction the classes taught.... Over time that portion of the paper will be generated. After you complete a publication quality figure...figure. Writing a scientific manuscript is like generating a quilt with your lab mates, you add the individual...

...(2023) Nanobodies: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci 24:5994. https:...The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended...into the clinic as well. The first sdAb based therapeutic was approved in 2018, with many others in clinical...clinical trials (Jin, et al. 2023). In addition to therapeutics, there is a lot of excitement around using sdAbs... synthetic single-domain antibody libraries to generate new sdAbs. There has also been some interesting... (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable domains of heavy...their impact grow as new sdAbs become easier to generate and more widely adopted. So thank you to the camelids...

...and discovery The first generation of CRISPR could cut DNA, the second generation could do base editing,...specificity: when purified, it could be programmed to generate specific cuts on the RNA target. In addition, ...most common RNA targeting system, Cas13, the "collateral" activity of the enzyme can lead to off targets...RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using Cas7-11   Use ...editing, and with the third generation came Prime editing — the ability to now make any small changes, including...small deletions and small insertions, leaving a several kilobase-sized gap in the gene editing field. The...precision into a genome, can be considered the fourth generation of CRISPR, one which drops itself neatly into...

...and the moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping...lists of forums on particular topics. Volunteer moderators set the rules for these forums and try to keep...questions! 2. The r/Science subreddit is well moderated. One of our original worries about sharing science...trolls (urban dictionary for trolling: The art of deliberately, cleverly, and secretly pissing people off, ...dialogue). Luckily r/Science has a great team of moderators who facilitated scheduling, filtered out inflammatory... sales-y in posts which falls in line with our general mission to share- as opposed to sell- science. ...expect. Fortunately, Reddit Science is heavily moderated. I started watching the questions and comments...

... Choosing a Cas9 for your knockout experiment Generating a no-fuss genetic knockout  Knock-out cells or...loads of sequences to select from - in many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA...targeting opposite strands must be available in moderate proximity to each other.  If having two sgRNAs...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...strand break.   Prime editors are the second generation of base modifiers available. This system utilizes...transcriptional activation domain - VP64.  Later generations of activators include the SunTag system which... The Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64...

...practiced or required by many other journals and several funding agencies (e.g. Wellcome Trust, NIH Data...information (or links to information) for assessment of veracity of the conclusions drawn. The main difference ...publication. Who is to say that the initial data-generating lab has efficiently trawled their own data? A...much time they had. It could be argued that data generators may, in light of the policy, hold onto data longer... available to everyone at once, including the generators. This practice doesn’t appear to harm the originating... as in an actual publication and not require interaction with the data authors. In some types of research...multiple animals on different occasions via video, generating huge amounts of data. When published, this data...

...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting... for sharing on the site. The entry form has 5 general question types and it takes approximately 15 minutes...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...virus? If you were using a genetic tool, did it operate as expected? If your experiment did not work, we...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...submission of an experimental image result that you generated with a caption. For microscopy images, please ...microscope used, and objective used. If you did not generate the image yourself and it is under copyright, ...

...high efficiency CRISPR-based approaches for the generation of knockins via homologous recombination with...approach is most well suited, since it routinely generates knockin efficiencies of 1-40% with G418-selected... CRISPR-based approach. Second, the AAV preps generally used for gene editing are low titre, and are therefore... most current CRISPR-based approaches for the generation of knockins, it is not always ideal. Because ...advantages of the AAV and CRISPR-based approaches, several groups have recently worked to enhance the efficiency...AAV and CRISPR-based approaches is described in several recent reviews (Howes and Schofield, 2015; Moser...PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating human somatic cell gene knockouts using recombinant...

...kits with greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of... by making it easy for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein...protein of interest. The plasmids used to generate these fusions all use the same restriction enzymes for...provided a solution for this problem by developing a general translation initiation sequence known as the Species-Independent...locus-specific libraries compatible with Illumina next generation sequencing in order to study evolutionary dynamics...These bridging primers are amplified and used to generate Illumina sequencing libraries using a 2-step PCR-based...open source platform and the libraries can be generated in a cost-effective manner with minimal bias. ...

...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector...Casey Maguire and colleagues from Massachusetts General Hospital and Harvard Medical School took a closer... antibodies. This is particularly useful for therapeutic applications in vivo where endogenous anti-AAV...anti-AAV antibodies often neutralize the therapeutic efficacy of AAV vectors. How are exosome AAVs produced...exosomes or stabilize their structure or their interaction with AAVs (Schiller et al., 2018). Exosomal vesicles...interest special contexts, such as the delivery of therapeutic proteins to the brain which requires crossing...cells using exosome-associated AAV." Molecular Therapy 25.2 (2017): 379-391. PubMed PMID: 28082074. PubMed...

...and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative diseases. The...Gateway-Compatible Toolbox is based on the original Tol2kit generated by the Chi-Bin Chien lab (Kwan et al., 2007) and... specific neuronal cell types involved in neurodegenerative diseases, the Cole Lab selected promoters ...The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced pluripotent stem cells...reprogramming somatic cells. A common method for generating iPSCs to express the transcription factors from...Polycistronic cassettes that contained KLF4s had overall diminished KLF4 expression levels; this altered...mScarlet. Bindels et al used a combination of deliberate and random mutagenesis to isolate the best RFP...

...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...protein, the repressor protein can bind to the operator. The bound repressor then prevents transcription... repressor (TetR) can bind to the tetracycline operator sequences (TetO), preventing transcription. In...have been engineered and improved. Takara’s “2nd generation Tet-Off vectors (Tet-Advanced)” are the most ...VP16. cTA binds to promoters containing putative operator sequences (CuO) (linked in groups of 6), allowing...the same manner, with LexA binding to the lexA operator (lexAop). Fusing the LexA DNA-binding domain (...refined the Q system and introduced the second generation QF2 activators. These activators solve the problem...

...piggyBac™ transposase or hyperactive transposase mutants [7], it is generally not as efficient as Cre ...How does the piggyBac™ system make it easier to generate transgenic cell lines? One of the major uses of... that have negative impacts on fitness. One consideration when making a transgenic cell line with the ...nucleases (CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits...completely seamless (i.e., so no other genetic alterations have occurred) and isolating the specific cells...the piggyBac™ system, these challenges can be considerably reduced. The piggyBac™ transposon system can...and M. H. Wilson, “piggyBac-ing models and new therapeutic strategies.,” Trends Biotechnol., vol. 33, no...

...produced from its serum several weeks later. Created with biorender.com. To generate polyclonal antibodies... response.   Polyclonal antibodies are generated by injecting an animal with an immunogen, then...might be the full-length protein that you want to generate antibodies against, or a unique peptide sequence...Freund’s adjuvant is injected, which stimulates the overall immune response. Once the initial antibody response...designed for experiments where antibodies raised in several different species are present. One common example...antibody for your experiment, you should either order several vials of the same lot of polyclonal antibody, or...experiments to be aware of signal strength. In general, polyclonal antibodies produce a higher level of...

...ion-channels and pumps in developmental biology, regeneration studies, and cancer biology. I was first introduced...patterning. Based upon trends in studies over the past several years, it seems that the more polarized (negative...  Additionally, researchers have reprogrammed regenerating tissues without using genome engineering. Planarian... briefly inhibited in the entire worm during regeneration, the worm creates two heads. Subsequent cutting...transcriptional or translational level. However, several genetic tools have been developed to address these...exciting emerging field, with many implications for regenerative medicine, evolution of body plans, synthetic...the advancement of research in birth defects, regeneration, medicine, and synthetic bioengineering. References...

...(tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack by both...effectively serving their function of guiding Cas9 to generate a targeted break. These advances in gRNA technology...foreign RNA. To protect the RNA from degradation, several simple methods have been employed which modify ...modification is best? Studies suggest coupling several of them together, for example phosphorothioates...more efficient on a budget is to simply swap out several of the ribonucleotides for deoxynucleotides. A ...A partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency...photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable 2-nitrobenzyl...

... with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system for C. elegans...    New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching Nexus (...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on...anti-GFP nanobody on the cell surface. When the cells interact, GFP is transferred to the receiver cell, and ...cell pairs.   Figure 2: Schematic of the interaction between the sender cell and the receiver cell...with BFP, sender cells expressing surface BFP generated BFP-labeled receiver cells. What’s more, G-baToN...provides versibility in tracking and modulating the interactions between cells.      Tang et al., eLife 2020...

... islandicus CRISPR-Cas system. 5. Phage therapy Phage therapy uses phage to treat bacterial infections...have discovered over 50 anti-CRISPR proteins that interact with CRISPR-Cas systems such as Cascade-Cas3, ... DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017), and AcrIIC3...2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes and prevents the correct...bacterial pathogens possess CRISPR-Cas systems, phage therapy might not be as effective. This is where Acr proteins...enough that they could be engineered to “armor” therapeutic phages to protect them from pathogenic bacteria...activity is an important step towards CRISPR-based therapies and for more precise editing in the lab. References...

... top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search engine ...Twitter into a paper-finding-machine. There are four general types of accounts you can follow: Scientists: ...updated. This Twitter list contains >300 Twitter literature bots, including a bot for biosensor papers, microbe...Semantic Scholar Semantic Scholar is an AI-backed literature search developed by the Allen Institute for Artificial...search terms. Key features Semantic Scholar has several ways to focus your search results. A bar graph ...quickly identify papers with greater influence, the average number of citations per year and the number of ...aren’t familiar with the topic.  Meta Meta is a literature discovery platform developed by the Chan Zuckerberg...

... to consider one of several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally...biology, the answer is always it depends, but generally the efficiency of the desired edit is reduced ...name implies (Nishimasu et. al, 2018). xCas9 was generated by the Liu lab through phage-assisted evolution...evolution, specifically with the goal of generating Cas enzymes with alternative PAM requirements (Zhao et. ...through structure-guided engineering of the PAM-interacting domain of Cas9. After multiple rounds of engineering...helps even when it’s not an ideal site: moving several bps away from a desired site does not have the ...most gene targeting experiments. Between SpRY and several of the other Cas variants described, nearly all...