...Epithelial Cells Generated from Patient iPSCs.,” Cell Rep., vol. 12, no. 9, pp. 1385–90, Aug. 2015. PubMed ...marker is included in a standard homology directed repair template to facilitate the selection of cells containing...donor is used by the cell’s host machinery for DNA repair by homologous recombination, which results in the... fact that PBx relies on the inverted terminal repeats (ITRs) found on the piggyBac™ donor for its activity... K. Schwarz, “Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted... with FLEx Vectors  Learn how Homology Directed Repair Is Used for Genome Editing Resources on Addgene.org...

...viral vectors: packaging capacity and the role of rep proteins in vector purity." Human gene therapy 18.2...rate limiting step and can immediately undergo replication and transcription. However, with scAAV the effective... complexes to study neural function. Often the reporter proteins associated with these complexes are so...cell populations. Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered... Vectors for Your Research Get Ready-to-Use AAV Preps Browse All of Our Viral Plasmids and Resources ...

... really great signs. The Congressman for Albany, Rep. Paul Tonko also came and spoke in support of science...Disclaimer: The views represented below are those of the writers and do not necessarily represent the views of ...for our public policies—many of which impact reproductive rights and justice. Whether it’s used to fight...misinformation and propaganda that limits access to full reproductive health care. And it’s needed most in low-income...According to the Guttmacher Institute, most women of reproductive age live in a state with at least two abortion...conflict with scientific evidence. We need representatives and policymakers who not only believe in the...

...CRMAGE: CRISPR Optimized MAGE Recombineering." Sci Rep. 6:19452. PubMed PMID: 26797514 Find plasmids from... by two CRISPR repeats. The spacer can be digested using BsaI, allowing a spacer-repeat “brick” to be ...include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease). Gateway ...plasmids, which contain various promoters and reporters, and subsequently inserted into a Cas9-containing...CRISPR arrays for dCas9-based transcriptional repression. The CRISPathBrick plasmid contains a nontargeting...were to develop an array using 3 distinct spacer-repeats, you could easily create 7 unique arrays (e.g. ...

...Optimized MAGE Recombineering. Sci Rep 6: . https://doi.org/10.1038/srep19452 Selle K, Fletcher JR, Tuson H...end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function...oligonucleotide repair template. Through recombineering, the locus of interest is modified to match the repair template...curing time. pCRISPomyces for editing in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive...elements, like a repair template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria ...success of Cas9 mediated repression in the Gram-positive bacterium, Streptococcus pneumoniae. Robert Husson...pTargetF contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each ...

... in the associated publication. Ma, et al. Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source...hard-to-study cell types. Natsume T, et al. Cell Rep. 2016. PubMed PMID: 27052166 Nishimura K, et al. ...native methyl-directed mismatch repair (MMR) system needs to be repressed in the host strain. To address...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief...posts for every great plasmid that comes into the repository, but be sure to let us know if you'd like to ...growing number of CRISPR libraries from Addgene’s repository that can be used to perform screens in human ...-ome using a quick, affordable, and ubiquitous reporter assay. In their recent publication, Kroeze et ...

...table, reprogramming efficiencies ranking as low, medium or high is based on the reprogramming efficiencies... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming. Additionally, lingering...rates of reprogramming when compared to MMLV-derived retroviruses, it is not as well repressed following...by terminal repeat sequences, which the PB transposase recognizes. Following reprogramming, transgenes...risk of lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with...RNA to reprogram cells is that it’s simple and efficient. It has a high efficiency of reprogramming when...

...Origin of Replication? See our Plasmids 101: Origin of Replication blog).  The replication problem Plasmids...directly inhibit replication by binding to the origin of replication and blocking replication machinery (del...inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated sequences within...increases, RepA transcription is repressed and replication is downregulated. In the second model, termed...cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part of the ...plasmid that contains the origin of replication and the replication control machinery (Need a refresher... with the same replicon are incompatible because they compete for the same replication control machinery...

...but browsing the 49 new preps available in the Addgene viral vector repository with a nice cup of hot ...viral prep with multiple serotypes pAAV-hSyn-GRAB_OTmut AAV1 Biosensor Yulong Li New viral prep pAAV-CAG-NIR-GECO2G...Fyhn New viral prep AAV-hSyn-Ribo-jGCaMP8m AAV1 Biosensor Marianne Fyhn New viral prep pAAV-Ef1a-fDIO... viral prep pAAV_BiSSTe10_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New viral prep pAAV_... viral prep pAAV_BiCHATe27_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New viral prep pAAV_BiCHATe27...New viral prep pAAV_BiVIPe4_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New viral prep pAAV_... viral prep pAAV_BiLAMP5e3_ChR2_mCherry AAV-PHPeB Optogenetics Gordon Fishell New viral prep pAAV_...

...fused to a streptavidin core that are able to retain reporter proteins fused to the streptavidin-binding ... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single ...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system...activation or repression. The TCS light sensors described in ACS Synthetic Biology (2014) represent the newest... from the reporter construct. The sgRNA targeting Gaussia luciferase can be easily replaced with an sgRNA...

...and reprogramming and direct reprogramming Explore different methods for delivery of reprogramming factors...prior to reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...valuable tool for regenerative medicine, direct reprogramming is an inefficient process, with the majority...patterns unique to the few cells that successfully reprogram. Unfortunately scRNA-seq only captures the gene...investigate how early expression patterns influence reprogramming outcomes. CellTag from the Morris Lab at Washington...linking ancestral gene expression patterns with reprogramming outcomes (Biddy et al., 2018). Tracking lineage...

... new preps available in the Addgene viral vector repository! This includes the first Addgene prep packaged...viral prep pAAV_hSyn-DIO-PdCO-mScarlet-WPRE AAV1, AAV5 Optogenetics Ofer Yizhar New viral prep with...Lin Tian New viral prep pAAV-hSyn-GRAB-gDA3m AAV1 Biosensors Yulong Li New viral prep pGP-AAV-GFAP-iGABASnFR2...part of the BRAIN Armamentarium Collection. These preps drive expression of either Cre recombinase or a ...New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor  Notes ...Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 Recombinases Esteban ... AAV PHP.eB Recombinases Jonathan Ting New viral prep AiP13779 - pAAV-AiE0779m_3xC2-minBG-iCre(R297T...

...required for replication and transcription (Figure 1). All viral genes are removed and replaced by a single...signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific gene start...

... used as a template for repair? Read on to find out! Homology-directed repair and genome engineering The...is to utilize the built-in repair pathway cells already have for repairing DNA double strand breaks – ...homology-directed repair (HDR) pathway in mammalian cells, but a less frequent DNA repair mechanism in general...suggestions. Disable the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR -...and tricks for harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding...a frameshift mutation. The majority of cellular repair events will generate these types of mutations, ...the cell to use the donor DNA as a template for repair, which will result in the incorporation of your...

... vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene repository each year...Cumulative AAV preps available at Addgene from 2016–2023 by type of tool. B) AAV preps distributed by ...AAV preps for the seven most popular serotypes available from Addgene from 2016–2023. B) AAV preps for...jGCaMP7, and jGCaMP8 viral preps distributed per year compared to other AAVs preps from 2016–2023. B) Inset...prioritizing which preps to include in our service allows us to make ready-to-use viral preps that are available...been finding and sharing stories unique to our repository and resources. As editor of Addgene’s eBooks ...both to produce and to verify the quality of the prep. This was a large factor in Addgene’s decision to...

...involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways... has a higher capacity for repair, as there is no requirement for a repair template (sister chromatid ...introduced breaks are repaired. The following factors are required for NHEJ repair regardless of end structure...introduced. Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated...accounting for less than 5% of repair events. However, the products of accurate repair are easily re-cleaved while...Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could... or extensive DNA synthesis. NHEJ also finishes repair of most types of breaks in tens of minutes – an...

... even more powerful following a report that one can use linear repair templates (ie. PCR-derived dsDNA...single-stranded repair templates (Farboud et al., 2019).   The Mello lab also reported that modifying ... 2014). A further development of this approach reported in 2017 uses the direct injection of a CRISPR/...Cas9 ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits ... I developed a co-selection approach relying on repair of a conditional-lethal mutation to identify edits...edits with minimal screening effort. Selecting for repair of a lethal mutation should remove much of the ...lethal mutation survive the selection; from a representative co-selection experiment, five knock-ins were...

...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...positives: Run replicates of the experiment to reduce the likelihood of indiscriminate reporter gene activation...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether...interacts with a transcriptional activator (1a) or a repressor (1b). Scientists took advantage of these discoveries...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI...

...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid ...of Klf4 did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon...SOX2 acts to reprogram human fibroblasts into induced pluripotent stem cells. By replacing wild type SOX2...the efficiency of iPSC reprogramming while the HP1 fusion eliminates reprogramming. These results argue ...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief...posts for every great plasmid that comes into the repository, but be sure to let us know if you'd like to ...KLF4, and c-MYC is the established model for reprogramming somatic cells. A common method for generating...

...of clones   lacIq lac repressor overproduced, expression from pLac repressed more   LacZ β-galactosidase...that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria...cloning, routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying to cut... dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying to cut... is essential for recombination and general DNA repair Reduces plasmid recombination, increases plasmid...subunit S12 of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline ...