...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...

...Like everyone else in the world, I (Wenning) was fascinated by CRISPR and rushed to test it. I still remember...the day, March 16, 2014, that I got my first preliminary results from a CRISPR genome editing experiment...was shared by many others around the world. Yes, finally, we have a technology, CRISPR, that is simple in...conventional gene targeting, the cost may be $50,000. Finally, compared to conventional methods of genome engineering...advice from John Doench’s gRNA design blog post. Finally, always remember that you are working with RNAs... active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32:1262–1267 . https://doi.org...in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun 9: . https...

...multicolor labeling is a powerful solution for discriminating between neurons for light microscopy-based ... introduced, stochasticity will result in a combinatorial expression of these three genes with different...inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible...overcome the limited expression levels of XFPs in the original method, we used the Tetracycline trans-activator... long, as too much XFP is toxic to the cells. Inadequate expression levels of XFPs - Reduce the amount...Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous...

... are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those without (non-LTR TEs)...contain two open reading frames (ORFs) which often terminate with a poly(A). ORF2 encodes endonuclease and ...transposase enzyme binds the transposon’s long terminal repeats (LTRs), induces a double-stranded break...synthetic transposable element developed from inactivated Tc1/mariner transposons found in fish (Sandoval-Villegas...it leaves behind the CAG DNA footprint from its terminal sequences at the excision site after cleavage ...medaka fish because its insertion into the fish’s tyrosinase gene caused albinism (Koga et al., 1996). Unlike...Tol2 or miniTol2 is a truncated version of the original Tol2 and has ~3-fold increase in transposition...

...protocols, including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor...can reliably detect single-base differences. The original creators of this technique visualized the ligation... have been generally found to have a higher discrimination at the upstream side of the ligation junction...including Taq DNA ligase, are naturally able to discriminate against ligating substrates containing base ... at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature... fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active...method allowed screening of all possible base combinations (Watson-Crick and mismatched) around the ligation...

...inventory of recombinases and recombinase-dependent vectors. We recently released several recombinases in various...Calcium sensors and GABA sensors Optogenetics Recombinases And more! Control AAV For many AAV experiments...densely-labeled neurons because background fluorescence from inactive neurons is reduced. This variant has an approximately...pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 (124651-AAV9) Recombinase AAV Targeting and manipulating specific, genetically-defined...

... Bryan Roth’s lab from the University of North Carolina, Connie Cepko’s lab from Harvard Medical School...provided valuable insight into AAV production. Finally, we are blessed to have an incredibly supportive...” Under the leadership of Addgene’s co-founder, Melina Fan, we created a new team to push this project...project involved every Addgenie in the company. Our Finance, Business and Legal teams had to pave the way for...export and shipping on dry ice. The Business and Finance Teams continue to facilitate the formation of partnerships...dedication to do this well ... because it did. So finally, a big thank you to the Addgenies who went the ...

...scientific support staff receives – in fact, the blog originally started as a way to easily store and share in-depth... everything goes through an editing process and final check before publication.   Do research! Nearly...that’s not enough, we consult with experts, watch webinars, or use other research methods. In the case of...to make sure absolutely everything was correct. Final touches We try to use figures and images to help...communicator "just sketch a western blot out quickly." Final design (right). Immediate and immense regret of ...image sharing repositories. Then we format, do a final review to catch those pesky typos, and hit publish...

...depending on the combination of fluorescent proteins expressed. CreLite-mediated recombination was found in...editing efficiencies in the mouse brain, liver, retina, heart, and skeletal muscle. The dosing required...cyanobacteria. This kit contains 96 parts including terminators and promoters, CRISPRi parts, selectable markers...longer present, the fusion proteins separate, inactivating Cre. This is critical for reducing Cre toxicity...various fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene...

...others you’ll encounter include monoclonal and recombinant antibodies). By the end, you’ll have a better...column and from the target protein. Pre-adsorption Finally, polyclonal antibodies may be pre-adsorbed to remove...batch variability. Once an animal has undergone a terminal bleed to acquire all of its serum, there is no...polyclonal antibody, or select a monoclonal or recombinant antibody instead. Otherwise, you might experience...of background noise compared to monoclonal or recombinant antibodies. This is because the polyclonal antibody...antibodies Learn about the advantages of using recombinant plasmid-based antibodies Resources on Addgene.org...

...generated with 4 different, binary 2in1 vectors with all possible combinations of tag orientation (NN, NC...activity.   With the addition of blue light, the terminal helices of the LOV2 domain unfold, disrupting ...responds to blue light quickly. Within minutes of inactivating the CASANOVA complex, fluorescent CRISPR complexes...the brain compute using electrical impulses to coordinate emotions, thoughts, and behavior. Historically..., where insertion of the GOI occurs through recombination. The 2in1 cloning system is well suited for...