...companies about the pricing and coverage of drugs, and developing internal operational capabilities. In just ...of combining targeted molecular therapies for cancer with chemotherapies, or developing innovative mobile...experts to work on “cases”. Cases present specific operational or commercial challenges or questions that clients...clinical research to discover and test the best new therapeutics. Consultants often work with such teams to help...human resources towards the development of a new therapy based on its market need and anticipated commercial...trained on how to work best with diverse teams, to leverage the preferences of different personality types... scientific background, training in reviewing literature, and critical analyses skills were applicable...

...first-generation bacterial ARGs (bARGs), used in bacteria, expressed poorly at body temperature (37oC)... transposase and outgrow the cells for a several generations to dilute the unintegrated plasmids (Fig....imaging was used. We set out to develop next-generation bARGs that could scatter ultrasound nonlinearly...Serratia sp. 39006. We next tested bARGSer with several inducible expression systems in E. coli. The arabinose-inducible...L-arabinose at 37oC for at least 12 hours, or at lower temperatures for longer. Cultures induced with arabinose ...  ARG expression in mammalian cells The first-generation mammalian ARGs (mARGs) enabled ultrasound visualization...in deep tissue. However, not unlike the first-generation bARGs, poor expression and acoustic properties...

...Wang et al., 2013, Cho et al., 2014). There are several variants of the CRISPR/Cas9 system floating around...efficacy in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post...to target that region. Or you may even wish to generate a point mutation and need to focus your attention...yourself for potential gRNA sites, or you can utilise several online tools that do it for you. One such option...selection, ordering the sgRNA is simple and easy. Several companies supply gRNA sequences, such as GenScript...validating your edit in mice once they have been generated (more on that later). Find Validated gRNAs to ... of EGFP. Using this technique, you can assess several sgRNA’s at the same time, and choose the most effective...

...more hands on While teaching courses about gene therapy and genetic manipulation, I and other professors...absorbed if they don’t get a chance apply it in an interactive setting. The problem is…how can you make a “...similar types of thinking go into developing gene therapy strategies for various diseases. That’s when I ...-on”, but as much as I try being compelling, interactive, and application-driven to boost curiosity, I...skills to apply those concepts and understand how therapeutic strategies work. Find Addgene Blog posts about...pick the best strategy and propose a final gene therapy approach for the disease to be openly discussed... this way, the students also had to practice transferable skills like team building, collaborative critical...

...gene therapy have also been in the limelight. The FDA approved the first oncolytic viral therapy in 2015... science research all the way to clinical gene therapy. But how can you use these amazing tools in your... how we understand what genes do! Viruses have several options to allow you to do exactly that: shRNAs...integration of a plasmid. Viral transduction is also generally less toxic to cells than transfections, preserving...libraries makes them the ideal vector for the job. Therapeutic applications Vaccine development Attenuated and...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including...being exploited as delivery mechanisms for gene therapy. The tractability of AAV, low immunogenicity, and...

...17). Federal funding for science has been decreasing for the last five years and the first federal budget...passion for photosynthetic communities, microbial interactions, and science education.  Disclaimer: The views... in education, government, and civilization in general (1). Science and politics? Science is an apolitical... human consumption in comparison to 37% of the general public (5, 6). Similarly 90% of active research...mostly due to human activity while much less of the general public agrees (5, 6). Many of the new administration...only be by advocating for science education and literacy that this changes.  2) Lumping all scientists ...marching to advocate for science education and literacy as I believe people that can see and understand...

...recipient interaction should be tested before being ruled out; there are many factors that interact with sex...strategic approaches to gender equality over the past several decades: “Fix the Numbers of Women” focuses on...wrong costs lives and money. According to the US General Accounting Office, between 1997 and 2000, ten drugs...analysis of the US Women’s Health Initiative Hormone Therapy Trial, for example, found that the study saved ...Reduced use of estrogen plus progestin hormone therapy resulted in 76,000 fewer cases of cardiovascular... find 263,000 more osteoporotic fractures. The overall economic savings to the US healthcare system was... cells and tissues, specifically in stem cell therapies. Let’s go back to why ten drugs were withdrawn...

...year and how these plasmids have been used to accelerate COVID-19 research in other labs. Spike protein... protein has had many opportunities to evolve. Several variants are now circulating and plasmids containing...from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding different spike...used these pseudoviruses to determine how effective sera from vaccinated individuals are at neutralizing ... the reverse transcriptase, protease, and Bst polymerase constructs from their assay to Addgene.  Drew...expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step RT-qPCR reaction...SARS-CoV-2 researchers to investigate how the virus interacts with human cells. Addgene’s mammalian target plasmids...

...researchers tested several scenarios from embryo development to myeloid proliferation in acute myeloid ... IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation and had ...particularly challenging as they are great in number, generally very small, and can contain different neurotransmitters...proteins coupled with Fibronectin intrabodies generated with mRNA display (FingRs), Xue Han’s lab has ...cell cycle attempt to understand development, regeneration, and certain disease states. However, it’s difficult...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3...withdrawn became entirely red as they could not proliferate. Eastman et al. Cell Reports 2020. https://doi.org...

...efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression...activation p300 acetyltransferase dCas9 fused to the catalytic domain of p300 acetyltransferase increases levels...transcription, also includes light-regulated histone methyltransferases and deacetylases. CRISPR and epigenetics ...initiates cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways...delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited...repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1...Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes spiroplasma...

...There are several facilities nationwide where you can obtain excellent quality, high titer rAAV (including...including Addgene!), but you can also generate rAAVs in your own lab with standard molecular biology tools...our ddPCR for AAV quantitation blog post.  We generally do not recommend using ELISA-based titering assays...inverted terminal repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089... of gene expression in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186...can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038/sj.gt...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...

...the functions of different genetic elements to generate non-natural tools such as novel biological circuits...and more. By combining these components, one can generate constructs to visualize cell cycle dynamics, titrate...applications. In one example, modular constructs generated with MXS-chaining were used to label subcellular..., MXS Chaining gave researchers the ability to generate the four initial unique constructs and later combine...ligation, the original pattern of the MCS is regenerated at the flanks of each ligation product, which...that the restriction sites of the MCS are only regenerated at the 5’ and 3’ termini of the ligation product...added to the 5’ or 3’ terminus using the newly generated MCS. MXS-chaining also has the advantage of being...

...providing many of the details not generally captured in the literature, scientists can use the Data Hub...commonly encountered in human gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org...Working with AAV vectors in the brain and peripheral nervous system presents a special challenge to scientists... process is often not detailed in the primary literature, and as a result can leave scientists under-informed...time and effort to test and optimize our vector - overall, I spent two years testing and reformulating our...dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https://doi.org/10.1038/s41434... Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https://doi.org/10.1089/hum.2009.169...

...FLEx cassette using next-generation sequencing (NGS) (Guerin, K. 2020). In general, we observe that 0.1 -... in the Central and Peripheral Nervous System and Relevance to Human Gene Therapy. Front Mol Neurosci ...FLEx Vectors Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...Vectors). While this strategy greatly reduced the overall level of off-target transgene expression, low levels...greater off-target expression just due to more overall viral particles getting into those cells. Therefore...transduces your cells of interest. We recommend testing several different serotypes to determine which one is the...course experiment to check expression levels over several weeks allows the researcher to determine when the...

...begin to use CRISPR as a therapy. Low frequency off-target effects may generate irreproducible data in ... CRISPR experiments, many are turning to next-generation sequencing options over older assays. Biased... search criteria are not exhaustive. Mismatch tolerance settings are often limited to off-target sites...sites of <4 bp. The off-target list is also generally weighted by the position of the mismatch along the ...CRISPR editing in the model cell line or organism. Several options exist for unbiased off-target detection...labeling, enrichment on streptavidin and next-generation sequencing (BLESS) (Crosetto et al. 2013). GUIDE-seq...consistent set of standards as the field seeks to generate reproducible, quality data on the role of genetic...

...plasmids. Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium ...survive within cells. The replicons of plasmids are generally different from the those used to replicate the... to make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...few copies depending on how they are regulated. Generally, control of replication is referred to as "relaxed...exclusively in E. coli? Gram negative bacteria in general? Both Gram negatives and Gram positives? Will you...compatibility with one or more other plasmids. Generally speaking, plasmids with the same ori are incompatible...

...structural studies of E3 ligase cereblon By Mike Lacy Several types of drugs target the ubiquitin E3 ligase cereblon...Lon and TBD domains, part of the HB domain, and several mutations to improve solubility and stability (...and mScarlet-I3, Oliver Hobert’s lab recently generated new constructs specifically for C. elegans (Cao...showed greater photostability and also matured considerably faster, with expression levels comparable to...notorious for its high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for...mechanosensor called PILATeS, Pulling-Induced Luciferase Association Tension Sensor (Zhong et al., 2024...sensor STReTCh, the 11-amino acid BiT fragment of luciferase is embedded in the I10 domain of the human titin...

...microglia The REVeRT Dual AAV Vector System Neurodegeneration Collection updates   Multiplexed perturbation...2024 under a CC-BY-NC-ND license. In this iteration, the size of the lentiviral insert was minimized...ensure this tRNA processing only occurs on the RNA polymerase III transcript, the sgRNAs were encoded on the...recombinant versions of mouse monoclonal antibodies generated and characterized by the Geiss Lab in their effort...A122D mutation in the rhodopsin domain conferred several new improvements. The new variant has increased...valuable tool for microglia research and broader therapeutic applications.  Find microglia-targeting AAV plasmids...10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav Kawde...

... We’ve previously discussed some of the general considerations for performing behavioral tests, but now... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...field box or a circular corridor apparatus using a camera or tracking software. Locomotion is a highly complex... of individual neurons and in terms of their interactions with one another. In addition to the neuronal...controlling muscle activity and coordinating movement, several additional brain areas that reflect motivational...sensitization and might open new ways for a novel therapeutic approach against drug addiction. Anxiety tests...rodents can be used to study anxiety levels in general, or anxiety associated with psychiatric diseases...

...One of the biggest barriers to gene therapy treatment of central nervous system (CNS) diseases is the...because in vivo transduction of AAV serotypes generally tracks between strains of mice. This suggests ... engineered PHP.B. PHP.B was created by first generating a diverse library of AAV9 mutants that had a ...inheritance pattern of PHP.B permissiveness. The first generation of outbred mice (F1) had half the level of CNS...transduction as their C57BL/6J parent. The second generation (F2) of outbred mice also showed a Mendelian ...galactose and AAVR to transduce cells. The Wilson Lab generated results similar to the Deverman Lab when they ...Transport across the Blood-Brain Barrier." Molecular Therapy 27.5 (2019): 912-921. PubMed PMID: 30819613. Hordeaux...