...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ...Nathaniel Roquet from the Lu Lab at MIT developed plasmid based systems that report the order in which specific... action. AAV: A Versatile Viral Tool for Gene Expression in Mammals Adeno-associated viruses (AAVs) are... plasmid...

...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...have two parts: (1) a genetically encoded “tag” expressed as a fusion with your favorite protein and (2)... Generate Fluorescent Protein Fusions Request Plasmids from the Michael Davidson Collection Find Fluorescent...photons. For example, moving from transient overexpression of protein fusions to gene-edited cells can...

...of the second plasmid can be done in one day in one assembly reaction. This second plasmid saves you a ...alerts when plasmids become available.  We’ve also had some requests for variations on the plasmids we’ve developed...and Myers for speaking with us! Find the Plasmids: Plasmids for protein tagging with CRISPR Additional... available at Addgene. For more details on the plasmids that are currently available along with detailed...several projects and this is one related to gene expression. One goal of the ENCODE project is to look at...system, what we are doing is adding in a second plasmid to target an epitope tag onto the transcription...second vector is a homology directed repair donor plasmid with homology arms and a selection marker. If things...

...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic... gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed them along with Cas9 in the T-cell line 2D10. ...of the LTRs joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV-1 reinfection...cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 .... For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, ...preventing re-infection would require sustained expression of Cas9/gRNAs, which could result in higher rates...the cells infected with HIV-1 only after stable expression of Cas9/gRNA. Viral production is higher in this...

...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...production. Whether redesigning viral packaging plasmids, investigating different viral production strategies...

... as a donor plasmid containing the transposon, along with a helper plasmid that expresses the transposase...lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...the dose of plasmid the cells receive over the whole reprogramming period and the plasmid will get diluted...larger plasmids have lower rates of transfection. Minicircles: Minicircles are like mini-plasmids. They...

...Browse fluorescent protein plasmids and resources See all Jaffrey lab plasmids ... cellular processes by transforming cells with plasmids encoding aptamer-tagged RNA and later introducing...aptamer downstream of a known promoter. Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment...aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...leading to conformational changes that control the expression of coding RNA. Spinach-based riboswitches can...SELEX, the RNA pool was cloned into bacterial expression vectors to create a library. Bacteria were transformed...quantify RNA transcription by RNA Polymerase III. By expressing Corn under promoters for transcripts uniquely...

...red light instead of blue to excite a molecule of GFP. Because red light has approximately half the energy...light, two photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point...infinitely small point of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot...Resources Online Microscopy Primer on the Molecular Expressions Website Other major microscopy manufacturers ... Find Empty Fluorescent Protein Backbones Find Plasmids for Optogenetics ...

...Turquoise (CeNL; Plasmid #85199), Yellow (YeNL; Plasmid #85201), Orange (OeNL; plasmid #85202) and Red ... Researchers have been sharing plasmids ever since there were plasmids to share. Back when I was in the...specific strategies and tactics for getting the plasmids he wanted. Addgene makes it so much easier to ...kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk...reporter was used to sort successfully infected/expressing cells by flow cytometry. The isolated cells were...fluorescent protein that could be co-imaged with GFP using the same wavelength for excitation. The result...CyOFP1-NLuc-CyOFP1; Antares). Direct injection of plasmids for Antares or firefly luciferase into mouse livers...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the... that restrict expression to this cell type. To this end, they constructed AAV plasmids bearing one of...bacterial and mammalian mCyRFP1 or CyRFP1 expression plasmids, FRET constructs, and CaMKII and RhoA sensors...

...the IP in parallel with both a sample known to express the protein of interest, such as a sample transiently...include knockout cell lines or tissues that do not express the protein. With this control set, you should ...In cases such as this, an epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest ...interactions of interest. Tagged proteins are typically expressed at much higher levels than endogenous proteins...antibody posts  Resources on Addgene.org: Find plasmids encoding recombinant antibodies at Addgene ...

...summer or just a bright new plasmid. One area with lots of bright new plasmids is our Fluorescent Biosensors...are generated by inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled...Figure 2: iGluSnFR3 displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR...SF-iGluSnFR. Image from Aggarwal 2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.... 2022, under CC-BY-NC license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI.... By fusing ITAM to an RTK of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence...et al. 2022, under CC-BY license.   Find pYtags plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal...

...markers were not increased compared to a control AAV-GFP. Not only did the AAV-SaCas9-sgRNA constructs mediate...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...activation. Split inteins have also been used to express base editors. References Chew WL, Tabebordbar ...HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: . https://doi.org/10.1038...bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71 . https://doi.org/10.1038...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...

...with mixed expression, with the expression measurable through an epitope tag such as FLAG/His/GFP. Fluorescent...epitopes. Detection of overexpressed (tagged or untagged) protein When expression plasmids are available, or...flow cytometry, and mRNA expression.   If your protein of interest is expressed at similar levels in ... involves the transient transfection of expression plasmids in a given cell line. For flow cytometry, ...differences between endogenous expression and transfected overexpression, this approach cannot confirm ...caused by overexpression. Additionally, if the cell line used has endogenous levels of expression, this can...preferred. The overexpression approach is therefore best suited to cell lines without any expression of the target...

...rewards could be substantial: Plasmid tRNA Synthetase Expression Host pMAH-POLY Polyspecific aminoacyl-tRNA...Resources on Addgene.org Find Plasmids for FRET Browse Empty Plasmids to Fluorescent Protein Fusions ...with natural chain termination at stop codons, expression levels of these tagged proteins are often very...oocytes, mainly). Nevertheless, tRNA suppression plasmids are available from depositing labs that have mastered...Fig. 1) which can be carried out using Addgene plasmids. His-tag labeling reagents You may already have...protein, permeabilized cells  Cy5NTAc 32 25-45 GFP (fluorescein is similar)   Lumio Red Tc: CCPGCC 48...

...Dangers of Using GFP for Protein Localization Resources on Addgene.org Find Plasmids for Mammalian Cells...along with Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal...

...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety... for a researcher to come into contact with than GFP. Similarly, if the viral vector carries an shRNA ...

...reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et...pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids for gRNA...site (MCS) between EGFP fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA...pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T... mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA." Scientific reports...fluorescent protein (EGFP) reconstitution. (a) Scheme of validation for DSB mediated EGFP expression cassette reconstitution...halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP. Using ...

...using a plasmid with multiple, individual expression cassettes, having the genes expressed from the same...genes. Plasmid Name Multicistronic Element Expression Type MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP... for each gene that is expressed, multicistronic vectors simultaneously express two or more separate proteins...cells that are expressing your gene of interest at high levels. Unlike vectors that express screenable or...multicistronic plasmids ensure that any cells that are positive for your marker should also be expressing your ...want to express multiple genes in the same cell. Although it is possible to drive co-expression by using... a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two...

...methylation in mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced... are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation...modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear....that help package DNA, were associated with gene expression or silencing. To alter DNA modifications, researchers...Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and...well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks...cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation...