...images. In the early days of structural biology, Byron Rubin, a crystallographer working with Jane Richardson...solution to this problem with the development of Byron’s Bender – a mechanical device that could be used...protein’s structure into experimentally determined electron density, have been known to say something silly... in the education community that All models are wrong….Some models are useful. While a physical model ...first Cas9 model is certainly useful, it is also wrong in at least one serious way. We represented the ...

... having already been immersed in an industry environment, have a better understanding of the fast, deadline-driven...up in an industry job, being immersed in that environment sooner rather than later can be helpful. Publishing...project might be challenging. In an academic environment, surrounded by lab members who are all working...Novartis, Biogen, Amgen, New England Biolabs, Regeneron, and Genentech, among others, and the list is ...opportunity to do some great science in a supportive environment. Both paths will give you myriad opportunities...

...interview is another way to make the virtual environment more comfortable and help you to keep yourself...institution, you can get a better sense of the environment of the department, the lab setting, and how students... can also be nerve wracking to ask questions in front of other prospective students; an online meeting...by talking to current students about the work environment and social aspects of the department, you may...Options Thanks to the much more developed virtual environment resulting from the pandemic, students who are...

... In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the sample ...image acquisition is performed using cameras with electron multiplying couple-charged device (EMCCD) or complementary...of transgenic mice in which a subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H ...LSFM with multiple tissue clearing strategies and neuronal labeling methods will greatly aid our understanding...331–336. Pubmed. P. Keller, M.B. Ahrens.  (2015) Neuron, 85, 462-483. Pubmed. E.H.K. Stelzer. (2015). Nature...

...cloning capacity of ~8 kb, so either a single polycistronic vector or multiple single-gene vectors can be...endogenous pluripotency genes. Retroviral vectors are prone to incomplete silencing of reprogramming transgenes...differentiation potential. Finally, retroviruses are prone to insertional mutagenesis and random integration...retroviral vectors. This makes it possible to use a polycistronic cassette that expresses multiple reprogramming...factors can be delivered either as a single polycistronic transgene or with four different adenoviruses... ~14.5kb minicircle expressing a 2A peptide polycistronic cassette comprised of OCT4, SOX2, LIN28, NANOG...

...dimeric green fluorescent protein (pdDronpa) (Zhou et al., 2018). pdDronpa dimers dissociate in response to... to cyan light (500nm). By fusing the two pdDronpa domains to the sites flanking the DNA-binding cleft...binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA binding and ...Cas9, dCas9 Gene editing Zhou et al., 2018   pdDronpa Cas9, dCas9 Gene editing, transcription Light-Dependent...photoactivatable transcription systems to induce neuronal differentiation. Nat Methods 14:963–966 . https...

...dependent on the photon budget and pushing the frontiers of fluorescence microscopy often requires more...easier and more functional in complex biological environments such as live cells and tissues (Figure 1). These...several others can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling...photon budget and allow us to push further the frontiers of biological imaging.  *Worried about your budget...Central PMCID: PMC4344395. 3. Lavis, Luke D., and Ronald T. Raines. "Bright ideas for chemical biology."...

...system that could do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1...experimental use. In 1996, Naldini et al. transduced rat neurons in vivo with beta-gal and other reporters, and ...vectors are incredibly popular in the lab - the Trono lab’s lentiviral packaging and envelope plasmids...Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. Science. 1996 Apr 12;272(5259):263-7. PubMed ...Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. J Virol. 1998 Nov;72(11):8463-71. ...

...myself in a technical support role only when I was confronted one day with a product issue in the lab. The ...the world easier. You should also demonstrate a strong willingness to help others with their problems,...sometimes comprised of several layers of support: A front-line team accepts any incoming requests and answers...because there are just so many things which can go wrong in a lengthy protocol. And customers may tell all...careers are open to experienced professionals with strong interest in the technical aspects of science and...

...virtue of liberating data from paper into an electronic environment and hence making it searchable and shareable...founder of Research Space. There are many types of electronic lab notebooks (ELNs), each with its own pros ... of Research Space, which provides the RSpace electronic lab notebook. Rory is based in Cambridge, Massachusetts...on the Addgene blog Find tips on choosing an electronic lab notebook Watch a livestream of the Minisymposium...

...CpG dinucleotides and CAC trinucleotides during neuronal maturation (Tillotson & Bird, 2019) and is a global...of symptoms in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy...therapy. In 2014, RSRT brought together a strong collaboration between two gene therapy labs (Brian Kaspar... half of the mutant MECP2 RNA in each of three neuronal subtypes of the hippocampus. This technology has...Modifications in Human-Induced Pluripotent Stem Cells. Front Genet 10: . https://doi.org/10.3389/fgene.2019.00625...

... increase diversity in the academic research environment. The “how many?” question lies at the base of... fact that training in the academic research environment is setup to prepare trainees for further work...receive as postdocs is not transferrable to other environments. Slide from Paula Stephan’s Talk (All slides...succeed in publishing in the current academic environment. Some great tips from this panel included:  ... the structural problems within the research environment itself. At Addgene we hope that all parties involved...

...aspects of research at National Aeronautics and Space Agency, the Environmental Protection Agency, and the ...Additionally, interest groups are often pandered to when strong or overlooked when small, but data is not going...Science face deep cuts in Trump’s first budget Environmental Science and Technology: Crossing The Imaginary...having evidence’ on Trump’s wiretap claim The Chronicle of Higher Education: “March for Science” Organizer...

...reviewer’s concerns, you are making your hypothesis stronger. When you share data at a conference poster session.... It can be extremely toxic in a lab or work environment if left unchecked. Substantive conflict occurs...handy: Empathy: If you often find yourself in confrontational situations with others because you simply cannot...Empathizing with people, especially people with whom you strongly disagree, is a critical component of conflict ...will be considered in the right, and one in the wrong. If you arbitrate, one party might get exactly what...

... and the linkage of intra- and extracellular environments. Found on nearly every cell in the human body...membrane, physically connecting the extracellular environment and the cytoskeleton. Most integrins have three...responses to mechanical forces in the cell’s microenvironment. The actin cytoskeleton exerts tensile force... by the ligand embedded in the extracellular environment. This further stabilizes the extended open integrin...integrin inhibitors and natural ligands, such as fibronectin, to block receptor function, e.g., cell adhesion...

... the midst of a rapidly changing, globalized environment re-emerging from international financial struggle...but also nurture a supportive and protective environment where individuals are empowered to productively...opportunities that truly exist outside their immediate frontiers. Moreover, sharing a culture/country of origin...work? IMP Pilot Program 2015-2015 Scholars need strong proactive mentors who have relevant expertise and...and entrepreneurial visions thereby opening new frontiers for young talent around the world. The wealth ...

...climate change to study plant and marine biology, environmental microbiology, or biofuel production. There’s...Research requires dedication and effort, so building a strong understanding of what you’re getting yourself into...all just people. I’ve found that in the right environment and with supportive mentors, being open and vulnerable... what I don’t know yet has helped me cultivate stronger relationships with my labmates. Sometimes what...the vital things to look for is a supportive environment—it makes all the difference in a lab experience...

...now available in Addgene’s repository for use in neurons, both in culture and in living brain tissue. “The...explained. “It helps us adapt to influences in our environments. We are able to react and learn. All of this... the LITEs to control gene expression in mouse neurons and in the brains of living mice both quickly and... want to apply LITEs for the transformation of neurons, she offers a tip: save yourself some time by making...

...fluorescent tags in neurons. Their toolkit named ORANGE (Open Resource for the Application of Neuronal Genome Editing...CRISPR/Cas9-based toolkit for epitope tagging in neurons Harold MacGillavry’s lab developed a CRISPR-based...targeted genomic integration of fluorescent tags in neurons.  Read the paper in bioRxiv Find the ORANGE toolkit...

...notebooks and binder on your desk. Arrange them in chronological order (or by project, if that works better for...can easily spot the colors and text. Tip 2: Use electronic to-do lists to access your tasks on the go Never...could easily lend one to another labmate? Though electronic lab notebooks (ELNs) have gained traction with...to borrow most frequently. Copy these into an electronic file sharing tool, such as Google Drive or Dropbox...

...Detection of Norepinephrine. Neuron, 102(4), 745-761.e8. https://doi.org/10.1016/j.neuron.2019.02.037 Kagiampaki... B, Representative images of HEK293T cells and neurons expressing nLightG or nLightR before/after application...dashed lines. Scale bars, 10 μm (HEK293T), 20 μm (neurons). Figure and caption adapted from Kagiampaki et...

...our lab’s environmental impact, but we can all do more to make research more environmentally friendly.... to address, but is often ignored in the lab environment. I recently attended the Go green! Innovative...institute! What should I do in the lab to help the environment? Well, you could: Collect the NEB and/or Millipore...

...potential as a scientist. But I expected something wrong. My capstone project was based on plasmid cloning...successful clones because the bands were all in the wrong place. Except for one. My manager speculated that...my very own research project! Coming from an environment with limited opportunities and support, puts ...puts into perspective just how much one’s environment changes them. Addgene gave me the chance to become ...

...transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being transcribed from...option described below, plasmids for making polycistronic gRNAs are also available from the Yang lab for...Plasmids: These plasmids are similar to the Csy4 polycistronic system described above, except that they use...gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs. Eukaryotic... Find plasmids from this paper at Addgene. 12. Ronda, Carlotta, et al. (2016). "CRMAGE: CRISPR Optimized...

...refresher if you ever lose track of which acronym is which. Acronym Full name Notes CRISPR terms gRNA... of CBEs and ABEs. This post includes a lot of acronyms, so we’ve included a glossary at the end if you...activity, and they are sometimes processed by error-prone non-homologous end joining (NHEJ). To mitigate this...edit. NHEJ Non-Homologous End Joining An error-prone cellular process to repair DSBs. Base Editing ...

...Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat 13: . https://doi.org/10.3389...different levels of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression...expression, while the human synapsin 1 promoter is neuronal specific (Haery et al., 2019). Most AAV transfer...

... paper in Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting ...Considerations For Precise Neuronal Targeting Using AAV Technologies Using AAV for neuronal tracing Resources...

...available with one sgRNA per operon or gene. The sgRNA pool can also target core operons in other pneumococcal...group optimized their initial system by using a stronger promoter and different biochemical secondary energy...AAV tools to selectively control parvalbumin interneurons from the Dimidschstein Lab More GCaMP8 ready-to-use...

...leaves patients incredibly sensitive to sunlight and prone to developing metastatic melanomas and other cancers...,” McSweeney said. “But, it is still sometimes wrongly diagnosed as a seizure disorder.” In 2012, researchers...sodium-potassium pump expressed specifically in neurons of the brain. McSweeney says they now think that...that mutations in the gene may prevent neurons from returning to basal conditions normally after periods...

...improved detection for tracking large populations of neurons in wide-field imaging (Dana et al., 2018). See ...Plasmid Serotype Name 119036  AAV1  pAAV-hsyn-flex-Voltron-ST DREADDs / Chemogenetics AAV We have also ...chemogenetic AAV for activating and inhibiting neurons. Minimally-tagged DREADD AAV from the Richie lab...with Cre for intersectional control of specific neuronal populations. We now offer a Flp-expressing AAV...

...photos of our pets. #SocialDistancing may send a wrong message for some people. We should recommend #PhysicalDistancing...the COVID-19 pandemic, check out Care For Your Coronavirus Anxiety and Taking Care of Your Behavioral Health...home, the STEM career exploration site Inventing Heron has a radio reporter challenge, and code.org has...necessarily to chat, but to mimic your usual work environment.  Be kind to yourself. This is a challenging ...

... chow, a different water filtration system, environmental stability in an incubator), but there’s a lot...for a transparent and collaborative research environment. Journals as enforcers Currently, most of the... formatting can feel like yet another hurdle in front of publication when all they really want to do is... of incentives. Right now, researchers are not strongly rewarded for being transparent in their communication...

...presented chronologically. With a resume however, most often people choose a reversed chronological style ...you did. Emphasize the skills. If you studied astronomy or bioinformatics data, then the chance is that...Communications Coordinator for the Department of Physics & Astronomy, University of British Columbia. Originally on...

...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and tet...pMD2.G. Find psPAX2. pMD2.G - Also from the Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid...generation lentiviral packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used...generation lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It also...

...flexibility for in vivo proximity labeling Increased neuronal expression using Cre-mediated AAV Targeted Evolution...flexibility for in vivo proximity labeling   By Ashley Waldron Proximity-dependent labeling has become a powerful...doi.org/10.1038/s41593-021-00969-4.   Increased neuronal expression using Cre-mediated AAV Targeted Evolution...found at least two variants that display increased neuronal expression in the brain while having decreased...

...The calcium is strong here….I can sense it….sound like a plot to an biology scifi movie? Or maybe it’...contraction, and calcium channels are activated for neuronal action potentials; it’s an ion of interest for...monitor calcium specifically in their favorite neurons. This specificity is feasible partially because...Neuromodulation: Engineering and Applications. Frontiers in Cellular Neuroscience. 2019 Oct Vol. 13; 1662...

...beginning. The well-characterized bacterial lac operon contains a gene called lacZ that encodes for the...the enzyme β-galactosidase. Expression of the lac operon is induced by lactose, and also by a lactose analogue...completely accurate, IPTG binds and inactivates the lac operon repressor, thereby allowing lac expression). When...in the medium to ensure transcription of the lac operon.   Tips for blue-white screening Use a good control...

...separated by 28 base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct...requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target... of gRNA cleavage.   Xie et. al constructed polycistronic glycine tRNA-gRNA genes (PTGs) in the forms ...Bacteria and viruses have illustrated the power of polycistronic gene organization, and its translation to CRISPR...

...the appropriate ligand - estradiol for hER or progesterone for hPR - leads to nuclear localization, binding...controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well as reporters...with various concentrations of estradiol and progesterone and showed that reporter expression is dependent...cognate inducer. Since induction via estradiol and progesterone do not affect each other, these systems can ...

...sex and gender analysis important? Doing research wrong costs lives and money. According to the US General...treated, and other hormonal, immunological, and environmental factors. How to include sex in experimental ...Science, Health & Medicine, Engineering, and Environment—a project aimed at harnessing the creative power...Evoloving Check Out Extracurricular Activities for a Strong Science Career Path Additional Resources Check...