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..., and electroporated them with an ssODN repair template in induced pluripotent stem cells made from the...fertilization. Since Ma et al. designed a repair template with two synonymous mutations to distinguish it...were able to distinguish between repair from the template vs repair from the maternal MYBPC3 allele. Interestingly...from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but instead from the mother...supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing through the NHEJ...NHEJ pathway. Created with BioRender.com. In later characterization studies, the edited embryos proceeded...embryos to undergo editing using the provided ssODN template. In fact, this finding led the authors to title...

...ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown within 1-2...methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein and the ...the target antibody works well. In larger cell populations, you can deliver these components together using... electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines... CRISPR/Cas9-based system to study epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9...-minute analysis of the kinetics of chromatin regulation and subsequent mathematical modeling. Its reversibility...amount of noise in reporter gene expression by modulating the induction of transcription factors 1 and ...

... in neurons reveals a mechanism for regulating its translatability." Science 343.6169 (2014): 419-422....Therefore, I set out to study how protein turnover is regulated in neurons. Protein turnover is the combined ...the abundance of proteins in cells. Homeostatic regulation of protein abundance is essential for normal ...acids. The use of radioactive materials is highly regulated, requires designated work areas and come with ...proper microscope setup we can observe the whole population of one particular protein in neurons or in any... has recently published techniques to follow translation in living single cells. However, before CRISPR-Cas9...synthesized proteins of interest were detected and later visualized by FUNCAT-PLA. Before these methods ...

...Because of the availability of tools for genetic manipulations, mice have become the model of choice for many...experimenter blinding to avoid bias Genetic manipulation in behavioral neuroscience using viral vectors... is often done in conjunction with a genetic manipulation so that behavior can be linked to a specific... region. When it comes to making the genetic manipulation, there are two main decisions to make: 1. Which...inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression levels of certain...spatial and temporal control of gene expression manipulation. They are injected using a stereotactic frame...depending on whether it is an overexpression gene, downregulation via RNAi or gene editing/deletion using CRISPR...

...and Sibernetic, a fluid mechanics simulator that can be used to simulate muscle tissue as it applies to ...of mRNA are important for post-transcriptional regulation in C. elegans. To study the role of 3’UTRs, the... C. elegans 3’UTRs structures, alternative polyadenylation for all protein coding mRNAs, and annotations...GAL-4 based bipartite expression system that can regulate gene expression in C.elegans. In this system a... to activation sequences upstream of genes to stimulate expression (Wang et al., 2016). In 2018, the lab...vectors that each contains a guide RNA and repair template of your choosing (Schwartz and Jorgensen, 2016...PMC3531189. Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans." Nucleic acids...

..., a researcher can cause them to interact by stimulation with blue light.  Similarly, light can also be... and is reversible upon termination of light stimulation. CRY2clust enables faster assembly and disassembly... and better distribution of clusters without accumulation in subcellular compartments such as nuclear ...Furthermore, CRY2clust, when integrated into a Ca2+ -modulating optogenetic system (OptoSTIM1), caused two-fold...Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool used by neuroscientists to...artifactual PSCs commonly observed with direct stimulation are largely absent in the soCoChR-expressing ...rescued by the xanthine produced via complete demethylation of caffeine. For example, the Barrick lab was...

...them in vivo and in vitro, especially in the mid-late 20th century with the advent of hybridoma technology...recombinantly append their proteins of interest with unrelated peptides that already had high quality antibodies...an amide group added to its C-terminus post-translationally, which turned out to be critical for antibody..., another group of researchers was working on isolating monoclonal antibodies to better probe the functions... c-Myc protein or the C-terminus, Evan, et al. isolated six different monoclonal antibodies (Evan, et ...sequenced the variable domains of Myc1-9E10 in the late 1990s (Fuchs, et al. 1997; Schiweck, et al. 1997...Evan GI, Lewis GK, Ramsay G, Bishop JM (1985) Isolation of monoclonal antibodies specific for human c-...

...popular tools because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they...research is the lack of a method for genetically manipulating neurons throughout the whole brain. Neurons ...Cre-expressing tissues. This inversion creates a template that’s suitable for PCR amplification using pre-designed...Cre in a cell type of interest, a scientist can isolate DNA from the infected animal and use the pre-designed...that cross the barrier can be identified by PCR later. The PNS presents a different problem: it’s spread...measured via GFP expression, was determined 3 weeks later. Table was derived from figure 2e in Chan et al....measured via GFP expression, was determined 3 weeks later. Table was derived from figure 3b in Chan et al....

...purified protein not always available Calculate relative quantification Normalizing to ubiquitously...variation. Confirmation of sequence can be done from relatively cheap plasmid sequencing. While rAbs are monoclonal...to run a western blot. It allows you to detect relatively low levels of antigen, while providing flexibility...assay design. Conjugated secondary antibodies are relatively cheap and easy to source, making it easy to switch...lower concentration of blocker, you can also use gelatin.  Protein-based blockers are inexpensive and easily...it to create a standard curve on your blot to calculate the amount in your sample. (Do ensure your blot... not available in a pure form, you can perform relative quantification, which measures protein amounts...

...have evolved over the years to keep up with the latest tools. In Addgene’s early days, we would ‘spot-...Falls sized rainbow   Over the years we have accumulated more than 300,000 individual nucleotide sequences...help us work in large formats, such as 96-well microplates, and thanks to our dedicated product and development...pipeline. After reviewing and testing several platforms for engineering pipelines, we selected Nextflow...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...running on a computer, so it safely operates in isolation, and can be easily shared with others without ...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

... base editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular...deaminates a target cytosine (C) into a uracil (U) base. Later the resultant U-G pair is either repaired by cellular...Transition mutation (C>T, G>A, A>G, and T>C) installation by base editors. Pathways that compete with ...editing efficiency based on the location of the ‘A’ relative to the protospacer adjacent motif (PAM). ABE7.10...edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018) (see CBE... They can, but they require other base editor platforms. Hua et al. constructed a number of plant base...editing experiments in plants Choose a base editing platform depending on the location of your target base ...

...causing mesothelioma many years later. While there is a dose-response relationship (the higher the exposure,...mesothelioma symptoms in their body. The average latency period (time from first asbestos exposure to the...and turned into Stage 3 or Stage 4 cancer. These later stages of a cancer diagnosis indicate, for Stage...another organ in the body. Such misdiagnosis and late discovery mean that most mesothelioma patients have...extremely poor prognosis. Together, the cause, long latency period, and rarity of mesothelioma make it different... Sadly, in many cases the cancer is diagnosed so late that these treatments do not work very well. The...drugs used to treat mesothelioma – pemetrexed and cisplatin –because it cuts off the blood supply to fast-...

...100,000 viral vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene repository...vectors that deliver GFP.   Figure 3: A) Cumulative AAV preps available at Addgene from 2016–2023...systemic capsid PHP.eB.   Figure 4: A) Cumulative AAV preps for the seven most popular serotypes.... We then added jGCaMP7 and eventually jGCaMP8, later generations of GCaMP6, to the service. While they...be a matter of timing, as jGCaMP7 was released relatively soon after GCaMP6. It could be a matter of the...Addgene launched our AAV Data Hub as a community platform for the sharing of that exact data. Here, requestors...develop new systems, new partnerships, and new platforms, from our early engagement with UPenn to our launch...

...the only means to target and manipulate specific cell types (populations) within neuronal networks in ...2003). Flex vectors revolutionize neuroscience Manipulation of specific cell types in neuronal networks ...living animals. To accomplish such cell specific manipulations in vivo, FLEx vectors expressing the light activatable...Atasoy et al. 2008; Cardin et al. 2009). Ligand regulated recombinase - keeping Cre out until needed But...time point? To allow temporal control, ligand regulated Cre and FLPe recombinase have been developed. ...recombinase expression while proteins needed for later experiments (cellular tracking for instance) can..., Colin, and A Francis Stewart. 1995. “Ligand-Regulated Site-Specific Recombination.” Proc. Natl. Acad...

... other molecular techniques to introduce time modulation of the TEV cleavage and improve utility of the...systems at Addgene! Schematic of the evolution platform to generate TEV protease variants. Catalysis by... transcription factor to enter the nucleus and modulate transcription of the mCitrine gene. mCherry is...pMAL-T-Avi-His/BirA, a vector commonly used for in vivo biotinylation via BirA and AviTAG. pMAK contains the signal...competent E.coli (NEB) using IPTG induction. To biotinylate the nanobodies, the authors used biotin dissolved...higher concentration, and results in uniform NB biotinylation. Once induced, the NBs could be easily purified...and gene activation (CRISPRa) are great ways to modulate the expression of nearly any gene via specific...

...inferred sequences present in your edited population and their relative representation in the edited pool. The...editing - enabling a ~100-fold reduction in cost relative to NGS-based amplicon sequencing. To use the tool...guide RNA sequence you used. The ICE tool will calculate overall editing efficiency and determine the profiles...different types of edits that are present and their relative abundances.   The ICE tool also provides a Knockout... DNA from both edited and unedited (control) populations is PCR-amplified and Sanger sequenced. ICE compares...during generation of the ICE Score, the Pearson correlation coefficient (r) is also computed and reported...distribution of indel sizes in the entire edited population of genomes. Hovering over each bar of the indel...

... restriction/methylation system. This means the strain can neither restrict nor methylate DNA. dcm: Similarly...inducible promoter systems (which will be discussed later). In addition to the basic genotypes outlined below...Similarly, strains with this mutation are unable to methylate cytosine within a particular sequence. Browse...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...of T7 RNAP from the genome; does not restrict methylated DNA fhuA2 lacZ::T7 gene1 [lon] ompT gal sulA11...recommended as these auxillary plasmids may be isolated during the prepping process. How does inducible...flavors—pLysS and pLysE—the difference being that the latter provides tighter control of basal expression. What...

...one AAV and a repair template in second a second AAV to deliver their repair template to mouse zygotes and...via AAV infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous...homologous recombination with a homologous repair template. It is this application - the creation of sequence-specific...allowing a researcher to fill it with the repair template. Bok and Porteus 2017 recently showed that by ... as RNPs and delivering two halves of a repair template spread across two AAVs, it is possible sequentially...this technique, Bok and Porteus deliver a repair template that would take up ~6.5 kB if delivered in a single...technique to efficiently deliver large repair templates for applications that require large gene fusions...

...point mutations. In addition to upregulating HDR, NHEJ must be downregulated to prevent the introduction ...short period of time, which would prevent the accumulation of unwanted mutations over time. DMD is a good...The homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping... when the genome editing frequency is low, the relative abundance of exon-skipped mRNA is high, likely...ataxia telangiectasia, congenital disorder of glycosylation, and Niemann-Pick disease type C. Although the...precision editing tailored to smaller patient populations. To bring DMD CRISPR therapy closer to the clinic...challenges above and more. One chief challenge is upregulating HDR in mature tissues, as this process is necessary...

...Method Notes Quantification of a process Plate-based assays (plate reader) Detect the average amount of ...of fluorescence across a population of cells (i.e. in a plate well). Use to track a process (e.g. enzyme...often used to monitor enzymatic activity or phosphorylation over time. You can also use biosensors to follow...transcriptional activation, conformational changes, translation, and protein translocation. Targeting your biosensor...fluorescent HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules ...