... While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges...systems such that it no longer requires the viral transactivator, Tat. For more information on the different...of concentration method depends of a variety of factors such as resources available, cost and downstream...determination of the packaging limit of lentiviral vectors. Hum Gene Ther. 2001 Oct 10;12(15):1893-905. PubMed...Tips for titering your lentivirus Visit the viral vector featured topic page Resources on Addgene.org ...Guide Find lentivirus plasmids Browse all viral vectors ...

...ask the depositing laboratory to review the discrepancies. If the depositing laboratory confirms that these...reference sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences... actin promoter, and rabbit beta-globin splice acceptor, contains sequence that is over 80% GC. Some IRES...information about our sequencing results in the Depositor Comments section at the bottom of the plasmid ...hope to make available as part of our OpenBio Repository sometime soon. Stay posted!     References ...

...engineered prime editors to expand their PAM flexibility. Find the engineered prime editors here A lentiviral...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...pubmed.ncbi.nlm.nih.gov/24390935/ Hypercompact genome editor CRISPR-Casɸ By Melina Fan The Doudna Lab adds to...discovery of CRISPR-Casɸ, a hypercompact genome editor from the Biggiephage clade of huge phages. In this... Scientists looking for more compact genome editors due to delivery constraints and scientists seeking...

...This guest post was contributed by Johnny Kung, Director of New Initiatives for the Personal Genetics Education...dialogue between scientists and communities. As the director of new initiatives at pgEd, I take on a variety...education and government; advising writers and directors in Hollywood; and working directly with community...sticky ethical dilemmas to dark episodes in the history of eugenics and research misconduct. Past ethical... in genetics research, their applications and regulatory policies; keeping tabs on the conversation about...implications of their work. Johnny Kung is the Director of New Initiatives for the Personal Genetics Education...

...Acr) proteins are phage-derived small protein inhibitors of CRISPR-Cas systems that help phages evade ...the best time to shut off Cas activity? Using inhibitor timing experiments, the Doudna lab found that ...also be used to reduce off-target edits of base editors, which convert a nucleotide base pair to another... means they can be delivered in vivo using AAV vectors. Having a way to shut off CRISPR-Cas systems prevents...Maxwell KL, Doudna JA (2017) A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell 170:1224-1233.e15 . https...Papayannopoulou T, Lieber A (2018) HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR...

...biosensor affinity for calcium.”  Primary Vector Type The vector type should describe how your plasmid ...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids ...Distribute pending QC. The name of the Principal Investigator and Organization where your plasmids were first...

...-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that...editing Hu et al. also engineered transcriptional activators using the xCas9 scaffold. With an NGG PAM, xCas9..., confirming the flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like...xCas9 3.7 fused to third generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing...a smaller increase in NGA editing. Adenine base editor xCas9 3.7-ABE also follows the same pattern, with...Plasmids for Mammalian Systems Learn about CRISPR History ...

... post was contributed by Laura Desrochers, a postdoctoral scientist at AstraZeneca. Why I chose an industry...time working on something extremely risky or exploratory. Getting a job afterwards: Fairly or unfairly...an industry setting. Scientific community and mentorship: As an industry postdoc, you might find yourself...project’s or a team’s, it is possible that your mentor (and even their department) could be laid off while...Desrochers! Laura Desrochers is currently a Postdoctoral Scientist at AstraZeneca. She has a background...blog posts here Find 9 tips for a successful postdoctoral experience Read Joanne Kamen's 8 tactics for...

...My lab's vector of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, ... a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too hot because...approach gives me a bit of flexibility since I can store the cell lysates at -80 °C for up to 6 months before... completed in one long day, or the virus can be stored at 4 °C and buffer exchanged the next day. Refer...Production of recombinant adeno-associated viral vectors for in vitro and in vivo use. Current protocols...tool for gene expression in mammals Use the PEI calculator for planning AAV packaging transfections Find...

...Enhancers are cis regulatory DNA sequences that, when bound by transcription factors, can increase a gene...CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method...2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original... three components: dCas9 with an added biotin acceptor site BirA, a biotin ligase a gRNA(s) targeting...patterns in chromatin interactions. The chromatin story told by CAPTURE 2.0 is important, but it’s not the...

...localization, dynamics, and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce...has gone into making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally ... protein fusions Transmembrane proteins, i.e. receptors and transporters, are integrated into lipid bilayers...spiderweb-like pattern of tubules can be grossly distorted into dense stacked membranes termed Organized ...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz... His interests include the quality control of secretory proteins in the Endoplasmic Reticulum and live...

...available by both the Jacob Corn and Alex Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An...This technique utilizes Cas9 proteins harboring receptor ligands (C in the figure above), which result ... potential complications, there are additional factors to take into account with these types of experiments...and purification, which will require additional laboratory reagents, equipment, and expertise. Final thoughts...production of the Cas9 protein and a gRNA in your laboratory, once these have been acquired, genomic editing...Central PMCID: PMC4289409. 3. Rouet, Romain, et al. "Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease ...

...production is Komagataella phaffii (a.k.a  Pichia pastoris) because it secretes fewer of its own proteins...using a desalting column or an ultrafiltration concentrator. As the name suggests, you may also concentrate... to their parental hybridomas. Aggregation and storage Aggregation of antibodies may impact their quality...testing. An aliquot of our recombinant antibodies is stored at 37 °C for a period of two weeks. After this ...we are also cautious during the aliquoting and storage process to prevent dropped tubes. Now that your...Varley P, Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture...

... (NHEJ) and alternative end joining (alt-EJ). Inhibitors to essential components of both pathways have...events (Yang et al., Int J Mol Sci). Adding these inhibitors to your culture media prior to and after Cas9...introduction increases HDR, just make sure to remove the inhibitors within a day or two to not affect the viability... one step further, you can inhibit cell cycle regulatory proteins responsible for the transition out of...to fuse an HR protein to the nuclease. Early HR factors fused to Cas9 have been shown to increase HDR frequency...committing a break to performing HR before other factors were loaded onto the break, potentially shuttling...

...small-molecule inhibitors of a particular PPI. In such experiments, small-molecule inhibitors are tested ...because FRET depends on a number of hard-to-achieve factors. For example, FRET requires that fusion proteins...signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of the first...between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more difficult...library to screen PPIs among 450 transcription factors in live fruit flies. Although BiFC open-reading...

...Our story today starts with a bit of Addgene history... As you may be aware, Melina Fan, her brother ...get Addgene from a concept to an up and running repository. As they built the foundation for what Addgene...previous years, this event was held at Executive Director Joanne Kamen’s home- a testament to the familial... confident that their teammates and project collaborators have completed their work thoroughly. It’s kind...Veckerelli is the Administrative and Operations Coordinator at Addgene. In addition to her operational responsibilities...

...yourself needing to brainstorm with a team. One major challenge with these brainstorming sessions is avoiding...with unlimited time? Another strategy for your brainstorm session is to start with something completely... ideas that I have found helpful in my team brainstorming sessions.  Allow the group time to think about...This is why perfectionists tend to also be procrastinators.  If you fall into this category, get out of...our lab space, our protocols and even our Lab Inventory Management system. I hope these tips help you ...

...bacterial world produce an assortment toxins to claim territory or kill competing microorganisms, but did you ...as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is...unmarked genetic modifications in the yeast Pichia pastoris. By combining both positive and counter selection...induced by methanol. Once linearized, the delivery vector can be integrated into the genome to introduce .../sok toxin-antitoxin system, they were able to monitor lacZ activity over 48 hours in vitro without antibiotic...marker for unmarked genetic modification of Pichia pastoris." FEMS yeast research 9.4 (2009): 600-609. PubMed...

...lysozyme to reduce basal expression levels; expression vector cannot have p15A origin of replication F- ompT...higher T7 lysozyme expression than pLysS; expression vector cannot have p15A origin of replication F- ompT...codons not normally used in E. coli. Expression vector cannot have p15A origin of replication F- ompT ...alleviates inclusion body formation. Expression vector cannot have p15A origin of replication fhuA2 ...Cis-repression of the E. coli T5 promoter (found on vectors such as pQE or similar), inducible under IPTG (...lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15...

...signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner Rho GTPase, leading...to reveal even more insights. Super-duper lipid editor And if recruiting proteins to the cell membrane...phosphatidic acid signaling (such as Hippo, AMPK and mTOR pathways) and to introduce unnatural head groups...membrane or lysosomes and its activity can be monitored by phosphatidic acid probe GFP-PASS. B) Time series...Additional resources on the Addgene blog Viral Vectors 101: Optogenetics Additional resources on Addgene.org...Activity-based directed evolution of a membrane editor in mammalian cells. Nature Chemistry, 15(7), 7....