...worked with high school students in the bay area to create a plasmid rap video for us (If you’re new to plasmids...unfortunate story of Rosalind Franklin and her incredibly important role in solving the structure of DNA...is a YouTuber and science educator looking for creative ways to implement the next generation science ...

...means to create barcoded gRNA libraries that can be used to combinatorially modify the genome, screen for ...revolutionized how we decode the genome by making it easy to create specific genetic perturbations. The ease with which... off-target activities have proven themselves incredibly useful for the efficient generation of individual...multiplexed CRISPR systems for high-throughput screening are vastly useful for mapping the combinatorial.... Hart, Traver, et al. "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer...Hiroko, et al. "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide...PMC4420636. 7. Ma, Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-...

...advantage of these discoveries to create a new system for screening protein-protein interactions. In later...identifying prey proteins after screening. It is also possible to screen libraries of bait and prey against...single-pass two-hybrid screening (6). See the Tips section for more on how to increase your rate of interactions...interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run replicates of ...cerevisiae in 1989 and are still used extensively to screen for molecular interactions in the cell, including...protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial studies...use many preys with a single bait in Y2H-based screens. In this way, proteins that interact with a molecule...

...transgenic models that express recombinases like Cre or Flp in their cells of interest and recombinase-dependent... pairs of specific sequences, lox (recognized by Cre) or frt (recognized by Flp), to either flip or excise...The two recombination sites are both recognized by Cre, but they are comprised of different sequences (loxp...lox2272) and can not recombine with each other. When Cre is introduced to the system it binds one pair of ...sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one...of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites, let's...to leaky expression in cells that do not express Cre or Flp. Here at Addgene we quantify the amount of...

...DIO. Lox sites are recognized by the recombinase Cre. Cre first flips the sequence between the first pair...them.  Lox sites Lox sites, in combination with the Cre recombinase, are used to spatially and temporally...flipped transgene. By restricting the availability of Cre, lox sites lets researchers control the spatial and... activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck Hepatitis Virus...which aids in nuclear export of mRNA and helps increase the expression of your transgene. The WPRE is ...

... vivo screens in naïve T cells. More broadly, this system can be used to perform in vivo screens in other...power of in vivo screens. Figure 1: Using FITS to perform in vivo CRISPR screens to assess T cell...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine...vivo. Over the past five years, in vivo CRISPR screens have been used to interrogate T cells, an important...complex and physiological microenvironments. These screens have primarily been performed in activated T cells...naïve T cells. The Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024...

... work more creative. An example could be making lab meetings more interactive and creating space for discussion...tools for creative interaction During Zoom meetings, different tools can be used to promote creative interaction...make the screen-sharing experience more interactive. The button is on the right of the Share Screen control...this is an underrated ritual that really drives creativity and novelty in scientific research and in any...Virtual lab meeting? No problem! You can still creatively collaborate and brainstorm! Image from Joseph...control panel on the top of the screen and every participant get to annotate things as they come up in their...their minds. The final screen can also be saved and exported as a picture. I believe that showing something...

... your selection, then screen Why add a screen to your cloning strategy? A screen will help you more easily...a look at a few screening methods. Blue-white screen A widely used method of screening is the blue-white...In many cases, you will need a screening strategy as well. In a screen, you aren’t killing off a portion...example on a plasmid, creates a functional 𝛽-galactosidase. Blue-white screens rely on vectors where ...blue-white screening blog post for more information.   Figure 1: Result of a blue-white screen. Image...game. By using a variety of screening and selection techniques, you can increase the chances of finding the...that contain your desired insert? This is where a  screen comes in. Let’s take a look at All_in_one_CRISPR...

...Cervical cancer screening in Ghana Gladys is currently working to enhance cervical cancer screening in Ghana....cervical cancer screening for a limited group of Ghanaian women by including PCR screening for HR HPV alongside...as positive controls in her PCR screens. She hopes that these screens will both identify individuals who...to be at high risk by these screens can then undergo more routine screening allowing for early cervical...positive controls in PCR-based cervical cancer screens. This third grant represents the continuation of...back in June, Gladys will be using plasmids in creative ways that we wouldn’t necessarily have expected...tests are commonly used in Ghana, there is little screening for DNA markers like the presence of high risk...

...payment for working more than 40 hours per week will increase from $23,660 to $47,476 per year, under updates...2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA, which had not seen...automatically adjusted every 3 years. In May 2016, Secretary of Labor Thomas Perez updated the overtime regulations...post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair Pay for Postdocs: ...made by multiple organizations across academia). Increased postdoc wages, overtime tracking? What are institutions...should therefore rise from $43,692 to $47,484, an increase of 8.7%. However, it is hard to gauge exactly ...negotiated a raise from $43,692 to $48,216, an increase of 10.4%. The Icahn School of Medicine at Mount...

...they need to use them in their own work, it also increases the visibility of both you and the original depositor...work later. We also hope that this feature will decrease redundancy. If you modify a plasmid from Addgene...out there Beyond these 3 big pushes, we try to increase your work’s visibility in a few other ways. Every...

... this technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle, Neville...to enable both negative and positive selection screening. They and their colleagues including Feng Zhang...at a time,” Wang said. “It was very difficult to screen in a pooled fashion or to go after thousands of...expression. The Yusa lab has used their library to screen for genes involved in bacterial toxin resistance... is now possible to conduct systematic genetic screens in mammalian cells,” David Sabatini, a senior author...Library Reference page.    References: Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang... (PubMed). Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian...

...drive expression of Cre-mCherry and a second promoter to drive expression of a Cre-dependent fluorescent...will only result in cells where Cre-mCherry is also expressed. If Cre-mCherry expresses in photoreceptors...photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells, only...horizontal cells transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters...different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of the start ...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific...

...AAV-GfaABC1D-Cre-4x6T AAV5 Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 ...Collection. These preps drive expression of either Cre recombinase or a fluorescent marker in the various...Recombinases Esteban Engel New serotype pAAV-hSyn-Cre-P2A-dTomato AAV PHP.eB Recombinases Rylan Larsen ...Larsen New serotype AiP13778 - pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) AAV PHP.eB Recombinases...viral prep AiP13779 - pAAV-AiE0779m_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN3779) AAV PHP.eB Recombinases... New viral prep AiP13781 - pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) AAV PHP.eB Recombinases...

...biological process. These screens provide an alternative to genome-wide RNAi screens, which although highly...similar pitfalls exist for CRISPR screens and how best to design these screens to controls for flaws. Recently...CRISPR knockout screen to analyze the following unanswered questions about CRISPR screen design. Unanswered...CRISPR/Cas9 screen design Are non-targeting guides an appropriate control? Many CRISPR screens use non-targeting...optimal conditions for CRISPR knockout screens, the Bassik lab created a human knockout library that has 10...library was validated via a growth screen and a ricin toxicity screen since essential and non-essential...The library performed well in both screens, with the growth screen having a 1% false discovery rate and...

...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited...gene activation in mind. “We had not managed to create a system to allow us to reliably activate essentially...the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate all...and make sure it’s working. You can only do that screen with a system you are really confident about working...her colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma ...CRISPR library enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here...validation of the top 10 hits.” Two-way CRISPR screening In October, Jonathan Weissman’s lab introduced...

...DreO-bGHpA 121675  AAV9  pAAV-EF1a-fDIO-Cre (FLP-dependent CRE) 55634  AAV1  pAAV-EF1a-mCherry-IRES-... constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-...membrane-embedding voltage detector. Find Archon1 and its cre-dependent version at Addgene! Fluorescent protein...

...these issues by creating immortal antibody-producing cell lines (hybridomas) that secrete antibodies in ...Neurotechnologies® (BRAIN) Initiative, will allow Addgene to create a much needed open-access recombinant antibody/...School of Medicine on this grant. Dr. Trimmer has created thousands of high-quality antibodies/nanobodies...reagent batch. To supplement this, Addgene will be creating a crowdsourced Data Hub (built off of the concept...resource to life,” says Dr. Trimmer. But in order to create such a resource, we need your help. You can participate... Director. “NABOR provides a new model for the creation and acquisition of affinity reagents and will ...funding laboratories, such as Dr. Trimmer’s lab, to create recombinant antibodies for use in neuroscience ...

...tested iTOP in a series of primary cell types using Cre and various loxP reporters. In both mouse and human...potential application of iTOP Having tested iTOP using Cre-lox recombination, D’Astolfo et al. were eager to...protein expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination. Both primary hESCs... cre-lox...

...researchers injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1...injected AAV2 particles into a Drd1-Cre transgenic mouse to deliver Cre-dependent expression of  Channelrhodopsin...-1 beta overexpression cassette. Expression of Cre in these neurons turns on overexpression of IL-1 beta...2014). Scientists from the University of Geneva (Creed et al., 2015) used optogenetics to support these...transmission from the mPFC projections to the NAc and decreased the locomotion of the animal. This confirms the...behavior in rats in the forced swim test as seen by increasing the ratio of time spent actively swimming versus...described here is only a small selection of the increasing number of studies using viral vectors in behavioral...

...Expression of hM4D(Gi)-mCherry from this virus is cre-dependent, meaning that once injected, the DREADD...DREADD will only be expressed in cells that express cre recombinase. Typically, this feature is used to attain...cell-specific expression in transgenic animals, where cre expression is restricted to a particular cell type...the first time, the Roth Lab recommends verifying cre expression prior to use. The lab additionally recommends...expressing cell lines, which can then be used both for screens (using pooled CRISPR knockout libraries) and individual...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and then...monoclonal Cas9-expression line prior to performing a screen or even a single gene knockout. Top AAV: pAAV-hSyn-DIO-hM4D...

... For example, a researcher can deliver Cre-dependent TVA to Cre-expressing neurons in a transgenic mouse...Addgene Blog AAVs CREATed for Gene Delivery to the CNS and PNS Advanced Uses of Cre-lox and Flp-FRT - ...viral protein EnvA on its surface will only infect Cre-positive, TVA-expressing cells (Osakada et al., 2013...

... the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from...were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell...) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with direct...

...Addgene depositors have developed heat shock-inducible Cre and Cas9 for easy genome engineering in species like... Plasmids 101: The Promoter Region Plasmids 101: Cre-lox Light-Switchable Two-Component Systems for E....Tetracycline (Tet) On Expression Systems at Addgene Cre-lox Guide and Plasmids     ...cell growth media with glucose decreases cAMP and represses pBad, decreasing promoter leakiness.   Other ...

...interneurons can be labelled with an AAV1 Cre virus combined with a Cre-dependent AAV9 tdTomato virus in the...learn more about the AAV Data Hub, and hear the incredible AAV talks given by Connie Cepko and Ian Wickersham...

...labeling Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) dCas9...-4.   Increased neuronal expression using Cre-mediated AAV Targeted Evolution method (M-CREATE) by: Brian...multiplexed version of the Cre-mediated AAV Targeted Evolution method (M-CREATE), in which one applies both...variants that display increased neuronal expression in the brain while having decreased expression in the ...zebrafish as a model organism, the Parton Lab recently created a flexible system that makes use of pre-existing...Image from Goertsen D, et al., 2022.  Find M-CREATE plasmids here!  Goertsen D, et al. Nature Neuroscience...

...control when you turn off expression for good, the Cre-Lox system offers inducible yet permanent gene excision...can be excised by intentional introduction of the Cre recombinase. This is a time-tested system but does...some permanent knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for ...!  Turn it on Turn it on forever Permanently increasing or turning on expression of a new protein is ... gRNA, to your gene of interest to temporarily increase expression. There are several flavors of activators...

...doxycycline, but it can also be regulated spatially with Cre-recombination. Read the paper in Frontiers in Molecular...Averof lab. It’s an alternative to the conventional Cre or Flp recombinases used to excise markers. It has...Cas12a inhibitors Using a bioinformatics and a screening approach, the Jennifer Doudna lab identified three...

...open-reading frame (DIO). Upon coexpression with cre recombinase (either from a mouse line or viral co-injection...type specificity dependent on the promoter of the cre recombinase while still taking advantage of the robust...an appropriate mouse line expressing a particular cre, viral expression of your opsin is no longer required...tracing with fluorescent proteins in nontransgenic and cre driver mice. Current protocols in neuroscience / ...: PMC4242517.  7. Madisen L, et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced... us to show causality in neural circuits with incredible temporal and spatial precision (I’m not going...optogenetics has already been proven. Therefore, a concrete, well defined, and robust behavioral output is...

...often made Cre-dependent. If Cre-dependent vectors are used in transgenic animals that express Cre in only...labelling the subset of infected cells that also express Cre. Another advantage of AAV vectors is that there are... traced neurons with an AAV9 vector expressing a Cre-dependent fluorescent marker. Injection into a conditional...conditional mutant mouse brain that expressed Cre recombinase only in pyramidal cells of the hippocampal...PMID: 4107258. Lo, L., and Anderson, D.J. (2011). A Cre-dependent, anterograde transsynaptic viral tracer...