...subset of cells. By labeling fewer cells in a population, it’s easier to visualize individual/non-overlapping...this were labor-intensive and required using manipulations such as injection of precise titrations of AAV... of Ca2+ are high in the cell, calcineurin dephosphorylates the protein CZI, which in turn binds to calcineurin-dependent...calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target calcineurin...activity measurements in stressed or aging cell populations. Figure 3: Micrographs of wild type (...al., 2019. Looking for an alternative to intercalating fluorescent dyes for DNA-protein interaction...tTALE-eGFP and tTALE-mCherry. In comparison to intercalating fluorescent dyes which can cause photo-induced...

...on all things HEK, from the first experiments isolating the cell line all the way to modern applications...applications. What the HEK is it?   HEK cells were first isolated in the 1970s by Alex van der Eb in the Netherlands...postdoc Frank Graham’s 293rd experiment, hence the nomenclature we use today: HEK-293. The exact origin of the... the fetal tissue which HEKs were isolated from is still a mystery, but it’s currently assumed that the...-day cell line is far removed from the initial isolate, it is difficult to determine its true tissue origin... to faithfully produce these proteins and its relatively low expression of native channels.  No matter...0995fje Tan, E., et al., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors...

...activity of a microtubule-associated protein to regulate cell growth dynamics Article contributed by Klaus... modified by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics with light by modifying EB1.... light - a finding which opens novel ways in manipulating and studying cellular dynamics. The photoinducible... faster than any genetic or pharmacological manipulations on cells, and has potential use for a wide range...Biology 2018 Pubmed PMID 29379139. The “Riboglow” platform for visualizing RNA production and localization...cobalamin binding riboswitches used in the Riboglow platform at Addgene. Braselmann E, et al. Nature Chemical...a new imaging-based bacterial colony screening platform, Oliver Griesbeck’s lab set out to engineer a ...

...for transcriptional regulation, so it requires a different approach for regulating transgene expression... generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail...systems currently lack the safety required for translational use. Viral Vectors Retroviral: Some of the...silencing of reprogramming transgenes and the up-regulation of corresponding endogenous pluripotency genes...Sano et al to learn how microRNAs can be used to regulate Sendai virus transgene expression. Transient .... The advantages of this method are that it’s relatively simple to implement and doesn’t require time-...the plasmid or silencing of the plasmid by DNA methylation. Another downside of using EBNA1 based vectors...

...to do: gene knockout, a specific base edit, or modulation of gene expression. Location and sequence are... into DNA. This also requires an exogenous DNA template. HDR, however, is a very low-efficiency process... is >30nt from the proximal ends of the repair template (Yang et al., 2013). This means that, for gene...the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander edits are possible if...inhibition by CRISPRa and CRISPRi Finally, for modulating gene expression at the level of transcription...possible when using knockout or transcriptional modulation approaches. Avoiding off-target effects The ...genetic screens, however, a library composed of the latter would likely be more effective, so long as care...

...repressible promoters regulated? Like inducible promoters, repressible promoters can be regulated via positive ... the strong yeast promoter pTEF). As ethanol accumulates, it binds to the repressor, enabling it to bind...promoter pADH1m, which maintains activity during the late ethanol consumption phase of yeast culture. Repressible...One common binary system is the GAL4/UAS system isolated from yeast. In this system, UAS basal promoter...respective UAS and lexAop sequences but are also regulated by QS and quinic acid. From Riabinia et. al. Using.... et al. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol ... and expanded Q-system reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed ...

...DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create ...sequencing to validate these constructs, as the relatively low cost and quick turn-around time of Sanger... sequential “pooled library prep”, in which a relatively large number of samples are subjected to a DNA-barcoding...samples individually, plexWell™ reduces every 96 well plate to a single tube after the first tagging step. As... samples (e.g. a collection of 96 plasmids on a plate) will typically have a wide range of input quantities... from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped to seqWell, where...

...attempt to create gene-edited babies. Several hours later, the scientist, Jiankiu He, confirmed that two such...editing techniques that are sufficiently safe to manipulate an embryo—even if it’s not necessarily via CRISPR...mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental groups...three branches – a private funder, government regulators, and an independent ethics council – all worked... process of good research practice: computer simulations, in vitro research, animal models, and several...question we are left with is not “what is the translational pathway for CRISPR/Cas9 research” but whether...stop punting decisions on scientific, ethical, regulatory, and general public consensus to the next meeting...

...consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses are varied... chromophore activates the response regulator via phosphorylation, promoting transcription of a GFP reporter...2005, when Tabor was working in Voigt's lab. Years later, these classic systems continue to be optimized ...the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription (see figure below)...spread across three plasmids, with the response regulator encoded in the genome. To make the system less...

...glycoproteins and other proteins that require post-translational modification may not be easily expressed in ... E. coli and S. cerevisiae expression vectors in late March. The first set of 383 constructs went online...control processes. For the many other COVID-19 related plasmids in our collection, we’ve relied on Sanger...first pass QC. The lab team manually transforms, innoculates, and performs DNA minipreps for small batches...scientists prepare Sanger reactions by mixing this isolated plasmid DNA with appropriate sequencing primers...mutation.  Once the samples arrive, each group of plates will have select samples prepared and screened ...intensive screening and the use of high throughput platforms, Addgene and Ginkgo are striking a balance by ...

... an unnatural amino acid in translation (Noren et al., 1989). In the late nineties, this was developed...factors can negatively impact both translation and fidelity of translation, leading to system-wide effects...which can help determine structure and function relationships or, in more practical applications, allow a ...non-interacting with) the cells’ own natural translation machinery; these are often incorporated into ... any of a huge variety of ncAAs to study the relationship between structure and function…if you can get...

...PCR amplify the template plasmid. The parental, nonmutated DNA is digested with methylated DNA-specific ... a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged...DNA-specific enzyme DpnI, and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair...repair. This method can work well, but poor template digestion can result in a high number of background...mutations, to amplify both strands of a plasmid template. In addition to containing the mutations, the ...After being treated with DpnI to digest away the template plasmid, these PCR products are directly transformed...

... target required physical isolation of the visually identified subpopulation. For traditional cell culture...nucleotides of double-stranded RNA (dsRNA) results in downregulation, or “silencing,” of any gene that contains ... through targeted mRNA degradation, blocking translation, or chromatin modifications of complementary ...hairpin RNA, or artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent method...silencing efficiency and labor-intensive processes to isolate RNAi plants. With a visual reporter of RNAi, phenotypes...culture, enriching your active RNAi subpopulation can be easily achieved through fluorescence-activated...culture can be harvested instead of microscopic isolation. The plasmids required to conduct an APT-based...

... diplomacy and international relations, advocacy, and writing legislation (see Figure 1 for career map... give advice and guidance to those that ask. Relationship building is key in science policy. Luckily, ...happy hours that you can attend to help build relationships.  Reach out to a science policy professional...can manifest via research and data gathering, regulatory efforts, policy implementation, program management... It occurs in a government setting: drafting legislation, making rules, and implementing the policy within...company who decides how they are going pursue regulatory approval for a new product or a well-endowed ...But some are also open to individuals from mid- to late-career backgrounds. In fact, many of my AAAS STPF...

...despite the manipulation. Additionally, not all sample types are amenable to genetic manipulation, nor can...your target then you would expect to see good correlation between the levels of target detected in both...both methods. These approaches have relatively low sensitivity, which can be problematic if you are trying...need to be aware of any possible alternative translation start sites or alternative splicing that could...also influence antibody binding by altering the relative concentration of potential antibody binders and... targeting a different epitope, and look for correlation between the results. Using antibodies against...cytometry using both antibodies, as well as a third unrelated antibody as a negative control (A). The two antibodies...

...particles or relatively short expression times, where as others, such as hSynapsin, are relatively weak and...sites and the open reading frame is inverted in relation to the promoter, hence the name, double-floxed...homologous pairs, and that the degree of homology is correlated with the level of spontaneous recombination. ... etc.) and even neuronal phenotype in a mixed population (Burger, C. 2004, Aschauer, DF. 2013, O’Carroll...differences in the expression of receptors, post-translational modifications, and other factors that AAV utilizes...this study were packaged at Addgene and had a relatively low recombination rate (0.1% and 0.13% respectively... Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated...

... and then stained 10 days later. Plaques are counted and titer is calculated in terms of plaque forming...medicine (2017).   1st Gen AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot...8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response against AdV. Importantly...low titer rAdV-S, and can be stored at -80C for later use or used immediately for amplification.*Pro-Tip...not use CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count...260nm corresponds to 1.1 x 1012 particles/mL. To calculate the particle number: OD260 reading x dilution ...

...the jobs they perform. This became foundational later on when we studied biological processes, like how...selectively infect a genetically defined cell population. While the EnvA genetic sequences are supplied...complex control of specifically defined neuronal populations. In one study, RABV monosynaptic tracing was ...neurons came from nearby neurons. It wasn’t until later time points that longer-range connections were seen...identify inputs to two major dopaminergic cell populations in two specific brain regions (Watabe-Uchida ...differences in the inputs to each of the two populations could be used to explain the differences in the... the functions of these two neuronal populations. Overall, this study laid the foundation for how different...

...activity or their translation disrupted through physical blocking of the translation machinery. In the...gene expression and functions of key regeneration-related genes in four inexpensive and commercially available...transcription to create dsRNA. Students can produce DNA templates via PCR off of the same plasmid and use these ...expression changes through time or after genetic manipulation. The technique involves using synthetically ...create synthetic riboprobes off of linearized DNA templates which they can easily generate off of a circular... was first discovered and characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching...

...specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9...The coding sequence for GFP is transcribed and translated along with the gene, producing both a tagged ...the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, we used ...human genes and will add more to the collection later this year. Designing the donor plasmid Figure... one thousand base pairs on each side, in fact. Later, when we transfect the cells, these large regions...percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with...using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag ...