...without worry, as I had never worked the traditional “9-5” job. Prior to this, the only work experience I had...Science Communication function at Addgene. Working a 9-5 As it turned out, Addgene uses something called “core...accustomed to the routine, I realized that the “9-5” was not as scary as it seemed. The transition into...intimidating to me, as I had a hard time thinking about how I could fill this time. I was relieved to find...attend to other commitments, without feeling guilty about missing work. As long as my work was finished on...

...It contains short promoter sequences at its 3’ and 5’ ends that are crucial cis-acting signals for genome...version of the viral genome. It contains the 3’ and 5’ ends of the genome which are required for replication...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated and...transcription: NP, VP35, VP30, and L (reviewed in 5). The RNA genome is encapsidated by the nucleoprotein...or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly ...PubMed PMID: 9971816. PubMed Central PMCID: PMC104478. 5. Mühlberger, Elke. "Filovirus replication and transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...

...spinal cord following injury. Gene Therapy, 26(5), Article 5. https://doi.org/10.1038/s41434-019-0075-6 Issa... Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different brain regions.  Image...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan...Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9), e76310...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Kanaan...Perspectives of AAV Tropism Modification. Pharmaceutics, 13(5), 750. https://doi.org/10.3390/pharmaceutics13050750... of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex...

...researchers. Assuming 5 hours of hands-on time to make each plasmid, we would have saved 5 million hours through...sharing. How much is 5 million hours? A colossal 547 years! While we are very excited about reaching this milestone...to picture (see the picture of our shipping room above), but as Joanne Kamens, Addgene’s executive director...

...your results. (Figure 5). The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, ...incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting ...and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not express CAs9 and are...cells transduced by the control GFP-gRNA (Figure 4, 5). When the gel was overexposed, I could even also ... the undigested control (Figure 4, lane 0, Figure 5, lane 1) is what confirms it’s a real digestion product...product and not another PCR product. Figure 5: gRNA-targeted BRAF sites were edited in A549 cells...the editing efficiency, and it did not (Figure 4). 5. In theory you could use densitometric analysis to...

...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to... could be the instability of the supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly...endonuclease) protein that can form a complex with a 5’-P-ssDNA guide at room temperature. Until that time...technology. To dig more into what researchers felt about NgAgo, I conducted a survey and asked researchers...successful, and the second section asked basic questions about the NgAgo protocols used by respondents (transfection...27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo: Genome-editing...

...disease, as well as those that play into late onset AD (5).     CRISPR/Cas9 corrects Alzheimer’s disease...Front. Oncol. https://doi.org/10.3389/fonc.2020.01387 5. Rohn et al. 2018. “The Potential of CRISPR/Cas9 Gene...Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (5 December 2013). 8. Gerald Schwank et al. "Functional... Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December 2013).    ...therapeutic.” There are many fundamental questions about CRISPR/Cas biology still left to answer as well,...

...crRNA DNA 5' NGG Specific blunt ended DSB in target DNA Cpf1 RuvC-like crRNA DNA 5' TTN Specific...Specific DSB in target DNA with 5' overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required...LwaCas13a is also amenable to multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown...28475872.  Find plasmids from this paper at Addgene.  5. Abudayyeh, Omar O., et al. “RNA targeting with CRISPR-Cas13...potential applications of Cas13a given what we know about its structure and function? For starters, Cas13a... Check Out Our CRISPR Featured Topic Page Learn about Anti-CRISPRs Multiplex Genome Editing with Cpf1 ...

...modifications, which can all potentially affect function [5]. We and others have reported that FP cysteine residues...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...FP-fusion proteins. Our studies and those of others (5, 6, 7, 10, 11) highlight the very real consequences...published oxidative optimized palette of FPs (moxFPs) [5]. The moxFPs overcome the inherent problems of popular...PMID: 23971632. Pubmed Central PMCID: PMC3806368. 5. Costantini, Lindsey M., et al. "A palette of fluorescent...moxFPs over standard FPs for the reasons listed above. In addition, because the moxFPs are highly monomeric...Medicine. Her doctoral research was conducted in the laboratory of Erik Lee Snapp and focused on optimizing fluorescent...

...Brandeis University. 5 easy steps to forming your own peer mentoring group Find 5-8 colleagues that you...these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8 month formal program...available to go around. My advice has been to find 5 interested peers and form your own peer mentoring ...for Scientists Series. I have been thinking a lot about Mentoring for over 10 years. Many successful scientists...Medical School researcher Connie Cepko to learn about her mentoring style. While working on this project...postdocs I have started to talk to trainees more about peer mentoring. They want to find mentors to help... questions I get is, “What are we going to talk about?” There are an infinite number of ideas on the internet...

...Methods, 20(6), 908–917. doi: 10.1038/s41592-023-01880-5. PMID: 37188954. Preprint available on bioRxiv – doi...activation induces cFos::GFP-iPAK4 at 3 or 6 h. Scale bar 5 µm. Images adapted from Lin et al., 2023 bioRxiv, ...intracellular recordings. Nature Biotechnology, 41(5), 631–639. doi: 10.1038/s41587-022-01524-7. PMID: ... slice (70 μm) immunohistochemistry (IHC) (Figure 5). PSD-95 is a scaffolding protein known for its roles... on their webpage: mAb3D Atlas.    Figure 5: Delipidated mouse brain tissue was stained with Anti-PSD...section of the Addgene antibody page, and learn more about the whole-brain reference atlas efforts, including...

...type targets. Comparatively, detection limits of about 5% mutant targets have been reported for mismatch...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...PMID: 25964300. PubMed Central PMCID: PMC4477672. 5. Sedlak, R. H. et al. Digital detection of endonuclease... triggered by engineered nucleases. G3 (Bethesda) 5, 407–415 (2015). PubMed PMID: 25566793. PubMed Central...advantages over mismatch nuclease assays that will be elaborated below. Digital PCR Digital PCR (dPCR) involves...

... our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency across different stem ...other suppliers with a typical turnaround time of 5-7 days. The crRNA and tracrRNA are annealed on a PCR...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using... in 96-well format using an Amaxa Shuttle device; 5 days after the transfection, the entire plate was ...commercial suppliers of CRISPR reagents (turnaround time 5-7 days); because the tracrRNA is the same in all csRNP...suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...J Biotechnol 241: 136-146. PubMed PMID: 27845164. 5. Clarke, Ryan, et al. "Enhanced bacterial immunity...

...MagIC-cryo-EM to determine several structures (at < 5 Å resolution) of the linker histone H1.8 in Xenopus...edited sequence anneal with each other, while the 5’ flaps containing the original DNA sequence anneal...anneal with each other. Excision of the annealed 5’ flaps yields an edited sequence with the attB/P landing...with twin prime editing. Nature Biotechnology, 40(5), 731–740. doi: https://doi.org/10.1038/s41587-021...8R] antibody shared by James Trimmer’s lab (Figure 5) as well as antibodies for identifying other cell ...anti-GFAP [N206A/8R] antibody here!   Figure 5: Free-floating rat brain sections stained with Anti-GFAP...transcriptional silencing (Nuñez et al., 2021). Now, in a collaboration with the Vallabh/Minikel Lab, the Weissman Lab...

...iPSCs from PBMCs. Most laboratories use Sendai virus to reprogram PBMCs efficiently [5]. However, the Sendai...establishment of virus-free hiPSCs usually require about 5-10 passages. Furthermore, working with Sendai virus...2003. 302(5644): p. 415-9. PubMed PMID: 14564000. 5. Seki, T., S. Yuasa, and K. Fukuda, Generation of ...pluripotent stem cells. Korean J Intern Med, 2014. 29(5): p. 547-57. PubMed PMID: 25228828. 7. Okita, K., ...p53-p21 pathway. Nature, 2009. 460(7259): p. 1132-5. PubMed  PMID: 19668191. PubMed Central PMCID: PMC2917235...source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of starting blood sample...

... Hive Beautifully filmed video about colony collapse disorder.     5. Plasmid Rap Plasmid rap video...Science with Tom. In this podcast, we learn more about Tom and pick his brain for advice on how to dive... Rap of Henrietta Lacks Students in Oakland rap about Henrietta Lacks - the African American woman whose...science standards and communicate science. Learn more about Tom on his youtube channel and at sciencewithtom.com...

... for 5 years and just recently stepped down. I have gained invaluable experience and even after 5 years...Addgene executive director Joanne Kamens has written a 5-part blog on the topic:  http://blog.addgene.org/management-for-scientists-managing-vs-leading...in as Chair of the Committee which I then led for 5 years.  Different leadership styles Being an effective...the work I agreed upon (just as I would in my laboratory work). Committee members have to work professionally...to work with many different types of people both above and below you. During my time at the NPA, there ...further opportunities opened up to me. As I spoke about my work with the NPA, I was ultimately invited to...society in my scientific field - ARVO (as mentioned above). I became an official member of the Committee and...

...Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear dsDNA starting from the 5’ end and...alternative approach is to flank the desired change with 4-5 silent changes in the wobble codons, i.e. make changes...changes to the third base pair of the adjacent 4-5 codons that alter the nucleotide sequence but not the... of the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview ...PMID: 19180090. PubMed Central PMCID: PMC2790811. 5. Thomason, L. C., Sawitzke, J. A., Li, X., Costantino...currently a research scientist in the Department of Laboratory Medicine at the University of Washington. She... Additional Resources on the Addgene Blog Read about Homology Directed Repair Use Knockout/Knockin Plasmids...

...proposal successful. Typical proposals consist of 3 to 5 aims, with secondary milestones to meet along the ...statements on the next round of applications. Step 5: Keep writing! Each written grant application builds...first. Additionally, reach out to peers and collaborators who have succeeded at grant writing to see what...

...retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et al...Anterograde transport has been observed for AAV1, -5, -8 (McFarland et al., 2009), and bidirectional transport...tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310....Improved Vectors for Gene Delivery. Curr Gene Ther 5(3), 299–310. PubMed PMID: 15975007. PubMed Central...Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed PMID: 19578477. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem... of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–206...