...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...

... 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...

... 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...

...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...

...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...

...cells that will be labeled in a given population is directly correlated to the length of the truncated attP...subset of cells. By labeling fewer cells in a population, it’s easier to visualize individual/non-overlapping...transgenic animals are commonly used to specifically manipulate a cell type of interest in an organism, all cells...strategy results in sparse labeling of the cell population that expresses SPARC and the fraction of cells...intermediate (I), or sparse (S) labeling of the cell population. Figure 2: Dense (c), intermediate (d),...Clandinin TR (2019) SPARC: a method to genetically manipulate precise proportions of cells. bioRxiv 788679;...

...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 ...

...completely digest the template DNA. Note—templates must have been isolated from methylation competent E. coli...and digest with methylation sensitive restriction enzyme DpnI to remove the templates. Digestion products...University of Connecticut. Molecular cloning is the isolation, insertion and amplification of a recombinant ...

...including histone acetylation by p300, histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1...HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating Gene Expression and CRISPR 101...notice! Our newest page in the Transcriptional Regulation category already has five blue flame plasmids... MQ1, and cytosine demethylation by Tet1. RNA Targeting: This page contains Cas13 constructs for mammalian...

...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...

...slight or large variations depending on how closely related these species are. If a gene was moved exactly ...exactly as is from one organism to a distantly related organism, the gene may not express well because of these...reference sequence database. Through a series of simulations and machine learning, they created a classifier...instances of rare codons, sometimes intentionally modulating expression in ways we don’t quite understand ...saw that a percent sequence identity below 85% correlates with synthetic sequences. The researchers then...growing trend of sourcing genes from distantly related organisms. In 2006, less than 1% of the genes deposited...deposited at Addgene were synthetic. But nine years later, over 20% of the genes deposited that year were ...

...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 ...

...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...

...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3, that’s ...the cytosol, this ratio is low; but when Keima accumulates in autolysosomes, this ratio is high. When untargeted...biosensor contains mRFP and GFP fused to the autophagy-related microtubule-associated proteins 1A/1B light chain...quenched in acidic environments, while mRFP is relatively stable and fluorescences even at the acidic pH...mRFP-GFP-LC3 Tandem Fluorescent Protein, but following translation, this fusion protein is cleaved to generate one... GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...autophagic flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...

...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...