...protein is composed of an n-terminal DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's...Splitting wild-type Cas9 into n-terminal (Cas9(N)-2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates...with different N- and C-terminal tags. Additionally, by using an adapted destination vector with a heterologous...bearing the "Bunch of Baby Spinach"aptamer, which is a brighter version of Spinach2, an RNA aptamer that fluoresces.... Bunch of Baby Spinach, or BoBS, consists of three tandem copies of the Baby Spinach core embedded in...with Broccoli Vegetable tags, including Spinach, Spinach2, and Broccoli, can benefit your RNA experiments... control of gene knockout or activation, the c-terminal cas9 fragment was fused with FK 506 binding protein...

... Other variants of Spinach include Baby Spinach, a miniaturized version of Spinach that retains similar... Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach. In doing so, they identified...was consequently brighter than Spinach. The initial studies using Spinach2 showed its ability to track RNA...imaging. Bunch of baby spinach, or BoBS, contains tandem repeats of Baby Spinach. It was developed in a...twice as bright as Spinach2 when bound to DFHBI. It’s also smaller than any form of Spinach - only 49 nucleotides...Addgene’s collection. Green fluorescent aptamers Spinach and Broccoli and their derivatives are aptamers..., but not quantitative, studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is...

...bacteria. In vivo, these recombination reactions are facilitated by the recombination of attachment sites ... in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the...or by Restriction Cloning Destination vector attR sites for recombination; ccdB gene for negative selection...vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene ...vitro version of the integration and excision recombination reactions that take place when lambda phage ...attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates...into the bacterial genome flanked by two new recombination sites (attL-left- and attR-right-, Figure 1)...

... primer on how Cre-lox recombination works. Advances in Cre-lox recombination have included inducible...coupling of Cre-lox with FLP-FRT recombination to generate additional recombination events, and the synthesis...excision results in one of three recombinations, and subsequent recombinations cannot occur. While the Brainbow...Brainbow-1.0 mice.) The combinatorial effect of multiple, independent recombination events leads to a rainbow...systems remain valuable to biology. Cre-lox recombination, discovered in the 1980s, is one of the most... loxP sites to specify distinct patterns of recombination. In 2007, Joshua Sanes and Jeff Lichtman, both...M-CFP (membrane tethered cyan). Without Cre recombination, RFP is expressed. Cre can mediate one of two...

...factor and promoter pairs referred to as binary systems. Binary systems are genetic tools that consist ...repressible element to this binary system (making this a repressible binary system). The GAL80 repressor...expression and largely defines the amount of recombinant protein that will be produced. Many commonly-...default ON to an OFF state, as well as repressible binary systems. In recent years, a number of more complex... previous post. In the past 30 years since the original Tet-Off system was described by Gossen and Bujard...inducible manner detailed in our previous post. While originally developed for use in mammalian cells, this system...consumption phase of yeast culture. Repressible Binary Systems GAL4/UAS In Drosophila or development studies...

...how the Cre-loxP recombination system can be used to induce site-specific recombination events, and that...sites to induce site specific recombination events: Cre recombinase binds loxP sites, while FLP binds ...presence of Cre recombinase.   Once the SSR of choice is introduced, recombination can proceed either...any additional recombination events are impossible even in the presence of Cre recombinase. This plasmid...BL. 2012. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression...orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment...such strategy--the FLEx switch--which utilizes recombination elements to turn off expression of one gene,...

...cytosine base editor by coupling a cytidine deaminase with the inactive dCas9 (Komor et al., 2016). These fusions...RNA adenine deaminases have been engineered and/or evolved to catalyze cytidine deamination instead of ...adenine deaminases. The team used directed evolution to create one from the RNA adenine deaminase TadA.   ...protein (catalytically dead or nickase) fused to a deaminase that makes the edit A gRNA targeting Cas to a ... and team wanted to force the cell to use the deaminated DNA strand as a template. To do so, they used...copying the base edit. Figure 2: Cytidine deamination takes place on the free strand of DNA and converts...created the Target-AID base editor using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida...

...protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase can be used alongside...only expressed following a recombination event. Tissue-specific Cre recombination in this mouse generates...may acquire one of a variety of colors when a combination of XFP are expressed. Each cell remains the same...with Brainbow The Lichtman group developed the original iteration of this technology in 2007. Used to ...cells will fluoresce either red or orange until recombination occurs. Following the development of Brainbow...the default state of the transgene such that recombination is necessary to excise the non-fluorescent YFP...that express the construct but do not undergo recombination can be detected by immunostaining of the truncated...

... Keep your contaminants away While it’s likely that you’re the main source of contamination for your experiments... their original condition and can replace your working stock if it gets too old or contaminated. This ...hopefully help you keep a well organized lab and contamination free environment for successful experiments:... up to half an hour can help keep your hood contamination free, but you should keep in mind that UV light...precautions necessary to keep your cells from becoming contaminated. Wipe down everything that’ll be brought under...entering the hood is another great way to prevent contamination. This sounds rather trivial, but often solves...is in progress.Learn more about cell culture contamination in this post from the ATCC Declutter! Keep the...

...for authentication and contamination) and cell lines can become cross contaminated and are invariably much...still used today to test for HeLa contamination. HeLa contamination is still a problem today. It is now...cell lines suffer from contamination with HeLa being the most frequent contaminant, closely followed by ...different researchers are free of contamination and match their original description. The oldest, largest...Unreproducible results are rife. Misidentified or cross-contaminated cells have been known about since 1966 when ...the majority in those days) were in fact all contaminated with HeLa cells, the first immortalized human...wasted in basic oncological research due to contamination are in the tens or hundreds of millions, let...

...machinery making an original C-G pair converted to T-A or reverted to the original C-G by base excision...invented CRISPR-base editors by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick...indel formation. After binding with DNA, the CBE deaminates a target cytosine (C) into a uracil (U) base...., there are no naturally known 'DNA adenosine deaminases' which would theoretically give rise to an adenine...resulted in the successful evolution of an adenosine deaminase that can act on DNA for adenine base editing. ...created a mutant library of E. coli tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed....          The artificially evolved adenosine deaminase catalyzes the conversion of a target ‘A’ to ‘I...

...site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then... MagIC-Cryo-EM for enrichment and structure determination LEA proteins protect samples during cryo-EM ...editing CHARM: A compact epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM...MagIC-Cryo-EM for sample enrichment and structure determination By Mike Lacy Conventional cryogenic electron ...MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic of workflow. B) Detail of spacer...Funabiki, H. (2024) MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules ...then the recombinase handles the installation of the donor sequence. The latest work evolves and engineers...

...phosphoserine into recombinant proteins, such as the kinase MEK1 (5). MEK1 is inactive without requisite...candidate regulatory kinase is known, it’s possible to generate non-phosphorylated recombinant protein and perform...in vitro kinase reactions. Often, however, researchers lack knowledge of the regulatory kinase, access ...access to active kinase for the reaction, or have difficulties with kinase specificity in vitro. The establishment...establishment of a system in which purified, recombinant protein could be produced with site-specific incorporation...incorporation of phosphoserine directly into recombinant proteins (5). Optimization of this system defined...create a pSer/phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic ...

...first segments of the spinal cord showing the cortico spinal tract, CST, spinal cord and cranial nerve...Cuneate tract, dCST= dorsal cortico spinal tract, vCST= ventral cortico spinal tract, dc= decussation of the...was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence optical sectioning...Spelman focused a laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected... lens oriented perpendicular to the plane of illumination (i.e. light sheet).  This is in contrast to ...described the single-plane or selective-plane illumination microscope (SPIM). In order to improve axial...optical sectioning of a specimen is based on discriminating the out-of-focus reflected light by using a...

...sites are eliminated by digestion/ligation and do not appear in the final construct. The destination vector...Type IIS enzyme and ligase. Although the original destination vector + insert may spontaneously religate...ligated following a heat inactivation step. If your genes of interest or destination vector contain multiple...correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning...complementary overhangs that direct assembly of the final ligation product. As shown below, a fragment with...overhangs. Entry DNA overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification...ligation can be done in one 30-minute reaction. The destination vector and entry vector(s) are placed in a single...

... or under certain stimuli. Cre recombinase catalyzes the recombination of DNA sequences located between.../c, and 129. Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized...Outbred strains of mice are useful for comparing dominant and recessive traits and their impact on diseases...or into embryonic stem cells. Non-homologous recombination incorporates this DNA randomly into the mouse...knockin strains are generated via homologous recombination. A targeting vector is electroporated into embryonic... ends to the breakpoint promotes homologous recombination of the targeting vector. CRISPR/Cas9-based systems... of small molecules that promote homologous recombination or the use of long single-stranded DNA have ...

...intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together ...modifications requires multiple recombination events to assemble the final plasmid. At least 5 fragments...assembly can be achieved in vivo using a bacterial recombination pathway that is present even in common lab cloning... The pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested...Garcia-Nafria et al., 2016) uses the bacterial recombination pathway to allow any cloning procedure to be...strain: when asking the cell to perform multiple recombination events, all linear fragments need to get into...For simple cloning requiring just one or two recombination events, homemade competent cells of 107 CFU/...

...ligated into Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt...Originally published Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang. CRISPR makes it easy...ordered assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden ...together to allow for ordered assembly into a destination vector that expresses Cas9. This is illustrated...drive ordered assembly into a Cas9-containing destination vector. Note: depending on which method you use...fewer than four gRNAs, you’ll clone in a polyT-termination sequence for each unused promoter. This step ...to generate all of the overhangs needed for the final ligation step. Plasmids are then digested using ...

...delivery By Cythina Arokiaraj (Gradinaru lab) Another useful viral tool deposited by the Gradinaru lab, AAV1...recommends these recombinant antibodies for use in western blots, though the original parent clone (M2...primate brains Endothelial-specific AAV delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome...mitochondrial membranes imaged with structured illumination microscopy in HeLa cells expressing COX8a-mStayGold... photostability and brightness compared to the original, though its maturation is even faster. Published...maintaining the bright and stable fluorescence of the original (Ivorra-Molla et al., 2023). They also named their...Hridaya Patel (Addgene co-op) Researchers in the Gradinaru lab have developed an engineered adeno-associated...

...though the data is heavily dominated by the U.S. (42%), Europe (23%), and China (14%). Spikes in plasmid...instabilities in the China data, so we recommend interpreting the model results for China with more uncertainty...entered lockdown for weeks to months, pausing or terminating employment for a significant fraction of the ...encountered COVID-19 at different times. Most notably, China was affected by COVID-19 earlier than the rest of...hierarchical model: Because the U.S., Europe, and China account for over 75% of all of our plasmid orders...COVID-19 event windows as 01/15/20 -- 03/15/20 for China and 03/11/20 -- 05/19/20 (the last date in our dataset...day across regions, with Europe most impacted and China least impacted. The inferred weekend effect is, ...