...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...

...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...

...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...

...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...

... 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...

...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...

...that stably express a gene of interest. While the general approach for using lentivirus, infect and select...consider is the age of your culture post thaw. In general, the higher the passage number of a culture the...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...parameters. For example, while freeze thaw is generally not recommended, Holic et al. found that lentivirus...your target cells Labs tend to follow the same general procedure for lentiviral infections, produce the...earliest steps in lentiviral transduction is the interaction between the viral envelop and the receptors on... on the target cell surface. The possible interactions and hence possible targets for a lentivirus are...

...in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. Most commercial antibodies...therefore, critical to understand how proteins interact with one another (or with DNA) to perform diverse...the most popular approaches to study protein interactions is the pull-down assay, wherein a protein of...analysed by mass spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin...however, to these IP-based assays is to find (or generate in-house) a pre-validated antibody for each target...throughput because numerous cell lines can be generated, each with a different tagged protein of interest...these factors prevent stem cell researchers from generating clonal knock-in lines, especially for ambitious...