...vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial constitutive or inducible...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can ...can be used to induce site-specific recombination events and is widely used in the field of mouse transgenics...(Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 under the control...deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. No time... higher titer of lentiviral particles than the original lentiCRISPR v1. Special note from the Zhang lab...

...neural connectivity (Nassi et al. 2015). The recombinant viruses typically used in neuroscience research...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in research...G receptor is restricted to presynaptic nerve terminals (Lafon 2005). Therefore, once RABV is released... it is only taken up by the presynaptic nerve terminals of adjacent neurons (i.e., it is taken up by projection... neural tracing. Using these technologies in combination with molecular tools (e.g., optogenetic or chemogenetic...characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study. J Gen Virol. ...Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors. Nat Protoc. 2013;8:1583...

...Miyawaki, Griesbeck, Heim, & Tsien, 1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying...tRNA suppression competes with natural chain termination at stop codons, expression levels of these tagged...acceptors, then biarsenicals are a good option. Originally developed by Roger Tsien and co-workers (Griffin...compounds such as British anti-Lewisite (which was originally developed to treat heavy metal intoxication in...insert your protein of interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition, since...so don’t be afraid to try novel, unpublished combinations of labeling strategies. FRET experimental design...Roger Y. Tsien. "Specific covalent labeling of recombinant protein molecules inside live cells." Science281.5374...

...guests, Melina Fan, Kenneth Fan, and Benjie Chen! Melina Fan is a co-founder of Addgene. Melina received... podcast, we sit down with Addgene’s Founders - Melina Fan, Ken Fan, and Benjie Chen. We learn what it...University, where she studied diabetes and metabolism. Melina has worked at Genetics Institute in Cambridge, ... led business efforts at Addgene as the Chief Financial Officer and Chief Operating Officer. Prior to ...

...Originally published Aug 30, 2018 and updated April 16, 2020. Sensitive and specific nucleic acid detection... diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment has become a... from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...detection by Cas13. This amplification step in combination with the ssRNA reporter enables SHERLOCK to detect..., Cox DBT, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV,...2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360...Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F,...

...not. The lack of an exemption for postdocs in the final ruling was, for a lot of us, frankly quite surprising...which leads to many complications, including the inability of some institutions to know exactly how many ...of 10.4%. The Icahn School of Medicine at Mount Sinai in New York is raising the minimum from $43,692 ... funding climate combined with the lack of a sustainable research enterprise, there is no easy way for...cheap trainee workforce has clearly not been a sustainable approach to biomedical research in the U.S. and...we must make real changes to make the system sustainable. For years, there have been recommendations that... they are necessary to produce a healthy and sustainable research ecosystem and that we cannot ignore ...

..., Bjorck 1984). Protein A, Protein G, or the recombinant Protein A/G are conjugated to agarose or magnetic...their primary antibody. To circumvent this, a recombinant protein, Protein A/G, was developed, which can...primary antibody and serum immunoglobulins indiscriminately, causing competition for binding sites. In...antibody will not co-elute with the target protein, eliminating the risk of the primary antibody interfering ...antibodies conjugated within the pores are often inaccessible to their targets. Magnetic beads are solid spheres...need to be verified using alternative methods.  Finally, IP can also be used to study proteins that interact...on Addgene.org: Find plasmids encoding recombinant antibodies at Addgene  ...

...host's replication machinery for elongation and termination. These types of plasmids tend to be high copy...synthesizes a new DNA strand at each replication fork. Termination sequences present in the chromosomal DNA ensure... although ATP does get hydrolyzed over time, inactivating the protein binding it (Grimwade et al., 2018...replication and balance population growth against sustainability. Plasmid DNA replication and regulation Plasmids...which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome. Since.... Thanks, stringent regulation! This post was originally written by Jason Niehaus in December 2015 and...Leonard, A. C. (2018). Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...to incorporating broad host range replicons, recombinant vectors can be modified further to increase the...cloning sites (Keen 1988).  Find broad host range recombinant vectors at Addgene!  This post was written by...

...three codons in eGFP to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202...reporter that monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time...downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair insertion...conversion to GFP after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (... Figure 3: Targeting pEF-BFP with a cytidine deaminase base editor results in shift in emission spectra...resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter...

...markers and homologous recombination As an alternative, endogenous homologous recombination enzymes can also...enzymes and those that use endogenous homologous recombination. Modifying bacterial genomes using exogenous...exchange requires two sequential homologous recombination events, but because these events are rare, each... ability to grow on sucrose, perhaps by sacB inactivation.  Bacterial toxins for counter-selection In ...mix. Introduction of the strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to ...genetic engineering in bacteria using homologous recombination." Current protocols in molecular biology 106.1...

... requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence... filter sets on a fluorescence microscope, and finally (iv) good toleration in fusion proteins. Since ... introducing two mutations (F64L, S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (... loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can bind loci in the...GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the alternative is to... subunit fusions that do not tolerate N- and C-terminal fusions (Adjobo-Hermans, 2011). Structural information...

...focus on defining these resources and not on the financial aspects. Money often carries an emotional weight...administrative services, managers, and techs’ financial books.  To find personnel salary ranges, go to...using animals, then that’s a cost that you can eliminate from your budget. Every cut you make signifies...signifies that you've made a conscious effort to eliminate some of the excessive costs in your lab. That in itself...specific aims. 2. Then make decisions based on financial values. All too often, we see early investigators...save you money in the short term. However, the financial risks associated with old equipment, like service.... Always focus on the science When negotiating finances, instead of solely discussing numerical values...

...single-stranded to double-stranded, and finally, drop once more during a final temperature increase. If there ...are cases in which it is an appropriate choice (Panina, 2018). Look for studies specifically aimed at ...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...M; Schwarz G; Pfaffl MW. 2003. Standardized determination of real-time PCR efficiency from a single reaction...Nucleic Acids Res. 31:e122.Thelwell N; Millington S; Solinas A; Booth J; Brown T. Mode of action and application..., Dong X, Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is ...11237753. Primer design resources PrimerBlastDINAmelMfoldBLASTEnsembl More resources on the Addgene blog...

...Executive Director, Joanne Kamens, and founders, Melina Fan, Ken Fan, and Benjie Chen. Last week, the Addgene...weren’t able to join us in person. Joanne Kamens, Melina Fan, and Ken Fan each spoke about what Addgene ...mentioned Addgene’s incredible growth and our determination to keep materials as affordable and accessible...without whom Addgene could not do what it does.  Melina talked about her grandfather’s humble beginnings... heartfelt portion of the entire night was when Melina and Ken called their parents up to the podium to... great (maybe too great!) time. Ten years ago, Melina Fan had an idea and her family helped bring it ...

...genetic tools: activators, inhibitors, sensors, recombinases, and more. With the help of adeno-associated...continuously refined and developed so there are many combinations to choose from for your experiments! Late last... aimed at brainstorming strategies for using combinations of AAV-based molecular tools for targeted neuronal...specific expression of molecular tools and sensors Combinations of classical genetic tools, for example Cre/...restricted light exposure to a specific area in combination with delivery of AAV at the injection site results...specificity of the targeted cell population.  Combinatorial strategies can also be used for retrograde tracing...

...these devastating afflictions. Elia Di Schiavi - Spinal muscular atrophy Dr. Elia Di Schiavi is a Research...Bioresources in Naples, Italy. His group works on spinal muscular atrophy, a disease in which disruption...of the smn1 gene results in the degeneration of spinal cord motor neurons. This disease is estimated to...While disruption of the smn1 gene is known to cause spinal muscular atrophy, the molecular mechanisms behind...clinic and thereby restore motor neuron function in spinal muscular atrophy patients. Noushin Nabavi - Peritoneal...Systematic review of incidence and prevalence of spinal muscular atrophy (SMA)." European Journal of Paediatric...

...CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers... really need to use FACS (like they did in the original publication). I thought I could see GFP expression...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%... cells at an MOI of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to...human line, using methods based on homologous recombination (Rago et al., 2007). Using CRISPR, I was able... active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Doench JG, Hartenian E, Graham DB, Tothova ...

...you are studying the effect of a new tau protein kinase inhibitor on slowing diseases progression in an...group of mice, the experimental group, with the new kinase inhibitor and a second group, the positive control...control group, with an alternative but well-studied kinase known to prevent tau hyperphosphorylation. You ...experimental protocol but does not receive either protein kinase inhibitor. Since the negative control group does...variability. If one were studying the effects of tau kinase inhibitors on mice, for example, it would be better...protocol and try to find a way to reduce, if not eliminate, the noise. This can be done either by modifying...labeling occurs (Burry 2011). Complex experiments Finally, additional controls may be required for more complex...

...expression.  Finally, the risks of potential toxicity and/or off-target effects (e.g. recombinase-dependent...rather than relying on other systems as proxies.  Finally, the size and receptor interactions of a serotype...Nonhuman Primates after Intramuscular Injection of Recombinant Adeno-Associated Virus. Journal of Virology, ...promoter results in robust transduction in the rat spinal cord following injury. Gene Therapy, 26(5), Article...adeno-associated viral vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy...doi.org/10.5607/en20006 Rolland, A.-S., Kareva, T., Yarygina, O., Kholodilov, N., & Burke, R. E. (2016). Expression...