...graduate program at the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors ...Although AAV2 can transduce many tissue types, it has low expression in several brain regions where other...serotypes to infect different cell types. In the CNS, it has been shown that there is variable transduction of...from the Original Location - Axonal Transport It has been demonstrated that certain serotypes can undergo... of movement, distance traveled, etc.) (4). This has also been identified with AAV2 (5), AAV6 (6) and ...required for the onset of gene expression. However, it has yet to be determined what gene expression looks like...problem in many experiments, a group of researchers has identified this feature of AAVs as a new tool for...

...studied but it is only the tip of the iceberg as it has been predicted that many others are still unknown...enzymes. In contrast to other Cas enzymes, Cas14 has not been found in bacterial genomes but only in the...add-on to a diagnostic toolkit, called DETECTR, which has been created by the authors. This kit uses Cas12 ... Cas systems, Jennifer Doudna’s lab analyzed metagenomic datasets to try to determine whether simpler ...they mined a database of microbial genome and metagenome from the Department of Energy’s Joint Genome ...

...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...The risk of generating RCL is low but extra care has to be taken especially if you are working with proto-oncogenes...previously used to drive expression from the LTRs) has been removed, Rev (which facilitates nuclear export...expression. In contrast, in the Tet-on system, tTA has been modified (and renamed rtTA) to bind TRE only...viral binding. This method, called spinoculation, has been found very effective for T cells transduction...to infect your cells of interest.   Advantages Disadvantages Can carry large transgenes (>8 kb) Potential... cells, it was obvious that its safety was a disadvantage particularly for clinical development. In addition...

... even society. One obvious way to do that is and has been to publish in journals with a high “impact factor...cited articles appearing in lower-prestige journals has risen steadily from 1995 to 2013. Considering citations...online access on scientific impact Partly, the story has to do with online accessibility. It is now about ...among the most cited. And, as scientific discourse has increasingly moved online, surely times have changed...articles, Anurag Acharya and colleagues report, the percentage of citations to articles in non-elite journals...

...also reduced.  Disadvantages Every rose has its thorns and every affinity reagent has its downsides. ...created in lab in situations where a suitable antibody has been identified but a smaller or less immunogenic...using combinatorial libraries, and high specificity has been reported for a number of targets. Due to their...use an alternative affinity reagent?  The main advantage of alternate affinity reagents is their size. ...nanostructures. Diabodies readily co-crystalize, a major advantage over antibodies (Kwon, 2019).  Single-domain antibodies...

...communities and welcome suggestions in the comments.  X has moved significantly away from the functionality and...conversation in the scientific community. Addgene has been considering a gradual move off X as a response...conflict with our values. For this reason, our timeline has accelerated. We significantly reduced posting on ...Bluesky: @addgene.bsky.social; or @addgene on Instagram, TikTok, Facebook, and LinkedIn. We are also exploring...

...10 min and then recheck the pH to ensure that it has not drifted. Filter the solution through a 0.22 µm...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Low serum...L-alanyl-L-glutamine (or stable alternative such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ...55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots...transfection to determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below...

...regulatory elements. To solve this problem, the Zhang lab has previously used SaCas9 (1053 aa/3.2 kb) with AAV ... these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...be the best option for HDR, but this possibility has not been experimentally confirmed. Researchers have...cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than ...2015). PubMed PMID: 26422227.   Potential advantages of Cpf1 Type II CRISPR systems based on Cas9 ...nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18...

...studying the genetic underpinnings of these processes has been complicated by the lack of precise genetic tools...Simplified in vivo delivery using SPH mice CRISPRa has largely been limited to in vitro settings due to ...can be delivered to the CNS by lentivirus, which has a larger packaging capacity than AAV. Such an approach... or coding regions. Typically, each gene isoform has a distinct transcriptional start site. Because CRISPRa...circumvented by the Yang Lab with the development of a Suntag-p65-HSF1 (SPH) transgenic mouse (Zhou et al., 2018...This mouse expresses a modified version of the “Suntag” Cas9 activator that recruits up to twenty copies...used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA overexpression...

...restriction enzyme, you will need to verify that it has be cloned in the correct orientation - this can be... A diagnostic restriction enzyme digest takes advantage of the fact that restriction enzymes cleave DNA...

...variant has increased signal-to-noise ratio and better ability to record subthreshold voltage fluctuations...optical pooled CRISPR screening, Paul Blainey’s lab has developed a multiplexing approach to Cas9-based pooled... stabilize the 3’ end of the pegRNA. As research has just begun with PE7, it will be exciting to see future...limitations of traditional AAVs, the Becirovic Lab has developed REVeRT, for gene delivery using reconstitution...Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator...-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia The...earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure 4), they determined that an A122D mutation...

...require heavy optimization. Claudia Vickers’s lab has designed novel SL fluorescent biosensors by integrating...showed that the receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high...recent months: Our selection of GCaMP8 AAV preps has expanded. Check out the new selection of ready-to-use...performing genetics studies in organoids. The Organoid EasyTag System is a straightforward workflow for doing ... Figure 1: Example schematic from the Organoid EasyTag system, showing the workflow for inducible CRISPR... Image from Sun et al. 2021. The Organoid EasyTag workflow combines thoughtfully designed plasmid...

...Show that as the story has progressed, you or your lab or science or your CV has grown as well. Find your...Task, Action, Result) interview technique, which has been addressed in much better pieces than I’m able... your structure. Develop your protagonist In most cases, the protagonist will be you. Make sure to highlight...enjoy, remember, and listen to narratives — take advantage of that in your work The part of my job I most...

...Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while GeoCas9 has maximum activity from 50...the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR...stability in human plasma The last trick GeoCas9 has up its sleeve is its increased longevity in human...stable when incubated in reaction buffer alone, but has limited activity when incubated in diluted human ...50-70 °C. The key advantage of GeoCas9 is that it functions at temperatures much warmer than SpCas9...

...It is fair to say that CRISPR has revolutionized molecular biology and has enabled functional studies in...enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these...knockout methods of the same gene. However, recent work has called this prevalent assumption into question. Specifically...multiple target sequences are concatenated. hpRNA has been extensively used in plant research and many ...constructs are available to the community. Each construct has unique features, such as inducible expression of ...tool requires careful balancing of the inherent advantages and limitations of any technique in the context...interest. Tools for RNAi Arguably the greatest advantage of RNAi is the straightforward nature of the technique—all...

...ElizabethKenkel) December 5, 2018 How social media has helped me in science It may have taken a bit of FOMO...Here are three examples of how using social media has helped me or my lab mates: For me, using social ...with social media as a scientist Using social media has enriched my professional development, and while it...helped you fit social media into your busy schedule. Has social media helped you advance your research or ...handle, but I wasn’t using them to my professional advantage. It wasn’t until I wrote a blog post about a research...start using social media to their professional advantage, I’ve selected five resources that have tips for...

...Service since 2009. He has  developed a proprietary, low cost cloning protocol that he has used for cloning ...least 24 bp with the template and if the sequence has high GC or AT content, increase the overlapping region... most of the troubleshooting regarding this step has to be strategy specific, there are few general parameters...ratio of 1:2 (plasmid:insert), but this parameter has to be optimized according to the strategy adopted...satellite colonies and they form when all the antibiotic has been used. Don’t worry too much, just pick the big...many softwares out there than can help you at this stage and that can be used to simulate in silico cloning...

...set as 1. Some proteins are very dim (e.g. TagRFP657, which has a brightness of 0.1) and this should be ...Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer ...protein Excitation & Emission (ex/em): Each FP has its unique ex/em peak. Therefore, choose FPs that...detect the emission. For example, if your microscope has only two lasers, at 488nm and 561nm, you will not...pHTomato (pH sensors), HyPer (H2O2 sensor), ArcLight (voltage sensor), and iGluSnFr (glutamate sensor). More ...can fold in <10min at 37°C, mCherry takes ~15min, TagRFP ~100min and DsRed ~10hours. Temperature: FPs maturation...

...reprogrammed less efficiently, no such correlation has been found between the age of the patients from whom...potentially promote tumorigenesis in vivo. The phenomenon has been recorded in past gene therapy trials and remains...reprogramming factors To date, the mRNA transfection method has not been used successfully to derive iPSCs from PBMCs...However, reprogramming of PBMCs by episomal vectors has yielded hiPSCs at a low efficiency (0.001-0.03%) ...efficiency: the pCXLE episomal vector toolkit It has been shown previously that suppression of TP53 enhances...hiPSCs than age-matched fibroblasts [3]. These advantages make PBMCs the ideal source of somatic cells ... This is to avoid the chances of insertional mutagenesis, which is the integration of transgenes into ...

...Recently, Xue Sherry Gao’s lab at Rice University has developed DAP arrays for efficient multiplex base...process and release its own gRNAs from a single array, has been a top choice for multiplex gene knockout or ...proper functions to enable efficient base editing and has yet to be developed for prime editing, both and their..., which is conserved in all living organisms and has previously been used for multiplex Cas9-nuclease ...lengthy promoters, such as U6 or EF1α promoters, has limited the number of gRNAs that can be packaged ...CRISPR tools, without compromising their strategic advantages in gene editing, while keeping the system to ...