...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, ...

...cytosine base editor by coupling a cytidine deaminase with the inactive dCas9 (Komor et al., 2016). These fusions...adenine deaminases.  They used directed evolution to create one from the RNA adenine deaminase TadA.   ...Originally published Aug 16, 2016 and last updated Aug 6, 2020 by Jennifer Tsang. When we talk about ...elements. Broadly: A Cas nickase or Cas fused to a deaminase that makes the edit A gRNA targeting Cas to a ...you’d need a way to force the cell to use the deaminated DNA strand as a template. To do so, the lab used...template, copying the base edit. 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...

...SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination. SLIC ...reading Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Li MZ, Elledge...only does this system not use site-specific recombination, it also doesn’t require a ligation step. Based...Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized... incomplete plasmid looks like a homologous recombination intermediate to bacteria, so it can be transformed...optionally boosts efficiency with the homologous recombination protein RecA. (It’s effective with as little... branch); or by incomplete PCR, which skips the final extension step to retain products with single-stranded...

...of extensive sequences used for cloning into the final translated products. Negative selection markers ...expression vectors additionally contain various combinations of protein tags (His, Avi, and GFP) as well ...blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen." Scientific...production of a downstream peptide, the transcription terminators in this series are just the tool you need. The...this portion of the BIOFAB Plasmid Set contain terminators of varying strengths centered within GFP/RFP ...characterized in Cambray et al., 2013. These terminators can be used to precisely tune your polycistronic... for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest. ...

... lambda sutures and subsequent determination of the proper coordinates of your brain region of interest...activation and the kinetics of opsin activity/inactivity. Dr. Deisseroth has also generously made many... as the vector core at the University of North Carolina) that offer his constructs. Importantly, control...open-reading frame (DIO). Upon coexpression with cre recombinase (either from a mouse line or viral co-injection...specificity dependent on the promoter of the cre recombinase while still taking advantage of the robust expression...identical for all of these surgeries. Identify coordinates through a rodent brain atlas or research papers...prior to using the virus. You may need to adjust coordinates based on your own method of identifying bregma...

...Agrobacterium binary vectors Vectors designed for use in Agrobacterium are termed binary vectors because...depositor Indu Maiti has created a new and versatile binary Ti vector for both transient and stable gene expression...and E. coli. Like plasmids used in other systems, binary vectors are cloned and prepped in E. coli. However...co-cultivation with the target plant species. Thus, binary vectors contain two origins of replication, one...transferred to the plant, similar to viral vectors’ terminal repeats. It’s a lot of information to fit into...into a single plasmid, so most first-generation binary vectors are quite large (>10 kb.) Since transformation...scientists want to transfer large genes, smaller binary vectors with a larger cloning capacity would be...

...the brain of Cre-expressing mice to trigger recombination and expression of a transgene (Lin et al., 2018...with precisely truncated attP sites to control recombination of the gene construct. A predictable number ...effector or retain the stop cassette following recombination. Image from Isaacman-Beck et al., 2019. Sparse... Sparse Predictive Activity through Recombinase Competition (SPARC), a genetic tool that allows for the... downstream gene expression. attP and attB, recombination sites. These sites are target sequences of PhiC31...where the stop cassette is removed during the recombination step, the effector is expressed. In cells where...expressed and the cells are not labeled. PhiC31, a recombinase. It specifically integrates an attP site into...

...natural cytidine deaminases, they evolved the adenine deaminase ADAR2DD to deaminate cytidine and used... dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase. Three rounds of rational ...Originally published Nov 30, 2017 and updated Jul 31, 2020. Cas13 enzymes are quickly becoming major ...editor: a Cas13 enzyme fused to an RNA adenosine deaminase (ADAR). Such a system would convert adenine to...protein, they examined ADAR1 and ADAR2, which deaminate adenosine to inosine in RNA, creating a functional...functional A->G change. They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA editing...the full ADAR2DD to further optimize cytidine deaminase activity. While RESCUE can perform C-to-U edits...

...of an AAV plasmid. Inverted terminal repeat (ITR) sequence Inverted terminal repeats (ITRs) are what makes...in them.  Lox sites Lox sites, in combination with the Cre recombinase, are used to spatially and temporally...loss, it’s best to propagate AAV constructs in recombination-deficient bacterial strains, such as Stbl3s....FLEX or DIO. Lox sites are recognized by the recombinase Cre. Cre first flips the sequence between the...excise the sequence between the second pair. The final result is a permanently flipped transgene. By restricting...the end of the AAV genome, just upstream of the final ITR.  polyA  The polyadenylation or polyA signal...the end of the AAV genome, just upstream of the final ITR and downstream of the WPRE. TL;DR? Watch our...

... 69090 pMJ915 MBP (N terminal); 6xHis (N terminal); 2xNLS (C terminal) 47327 pET-28b-Cas9-... 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis (C terminal); NLS (C terminal...62934 pET-NLS-Cas9-6xHis 6xHis (C terminal); NLS (N terminal)   Easi-CRISPR efficiency and delivery...marker, likely by breeding to a mouse line with a recombinase. If it turned out your chimeric mice did not ... a standard dsDNA repair template is used. The original design for creating a conditional allele, or floxed...that the T7 promoter will not be included in the final ssDNA template. Although effective, ivTRT may be...as fluorescent proteins (EGFP, mCherry etc.), recombinases (Cre, Flp etc.), and tetracycline drug induction...

...wtGFP is illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly to the ...activates and inactivates Dronpa is not ideal, since this means Dronpa is quickly inactivated and less photons...PA-GFP to display almost no fluorescence when illuminated with 488nm light. However, when UV-violet light... to a 100-fold increase in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists...derived from DsRed and mRFP, respectively. When illuminated with violet light, both variants are photoconverted...but then further exposure to blue light also inactivates the chromophore, leading to a non-fluorescent...find a version of Dronpa that doesn't become inactivated as quickly. Padron is a monomeric, reversible...

... “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector cells...interleukin (IL)-12 and interferon gamma (IFNγ) - and eliminate cancer cells. 2. Equilibrium - In a process analogous...in bacteria, the tumor cells that escape the elimination stage acquire genetic alterations and alter cell-surface...In contrast to the acute immune responses that eliminate early stage cancer cells, chronic inflammatory...tolerance-producing antigens enunciated above. Finally, signals released from the microenvironment help...cells, and often cannot eradicate metastasis or eliminate the probability of a relapse. Chemical and radiation...to aide chemotherapy and result in successful elimination of certain tumors. However, antibody stability...

...mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons. Science 295:2258–2261...generally accepted that some form of homologous recombination (HR) occurs via this region of overlap, but ...single over-sized vector, but not always.  The combination of serotype and tissue/cell type could have a...fragments are likely non-functional, they could be dominant-negative and/or toxic. A potential solution is...repairs Overview: The sequential homologous recombination approach delivers large donor templates using...Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting. Front Neurosci 11: . https://doi.org...Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger...