...and generate virus (right), similar to how natural viruses can be delivered to a host and generate virus... Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Curr Gene Ther. 2013 Dec;13...can direct long-term transgene expression, but generally are not permanently integrated into the host genome...oncolytic) adenoviruses are utilized for cancer therapy because of their ability to preferentially infect...can be used to target specific cells or tissues. Overall, viruses can efficiently get genetic material into...an organism. Figure 1. Using viral plasmids to generate virus. A) Viral plasmids have been engineered ...genetic material (termed the viral genome) that, in general, encodes for more viruses. When a virus infects...

...to measure protein-protein interactions; if two proteins of interest interact in a bacterial two-hybrid...pTac GFP, Luciferase (LuxCDABE) Attila Karsi Broad host range vectors for expressing luciferase and fluorescent...to a particular sequence? Do these two proteins interact? What happens when I overexpress this enzyme at... redirecting metabolic pathways to produce a therapeutic compound, there are many different ways to alter...appropriate construct, grown at the permissive temperature (32°C) to allow transposition, and the construct... then cured by growing at the non-permissive temperature (42°C). Transposition is efficient enough that...about your protein’s function, structure, and interactions! Plasmid ID Promoter Epitope Tag(s) ID Purpose...

...used with 2nd and 3rd generation lentivirus technologies. ID Plasmid Generation Type Description PI 12253...transgene expression. There are some safety considerations to evaluate when working with lentivirus: these...potential for insertional mutagensis. The 2nd and 3rd generation lentiviral systems available at Addgene have ...information about: Lentiviral components 2nd versus 3rd generation lentiviral systems Viral production Frequently...produce an infectious viral particle (a virion) are generally divided among multiple plasmids. Some plasmids... Packaging Contains Rev; can be used with 3rd generation transfer plasmids along with additional packaging...Packaging Contains Gag and Pol; can be used with 3rd generation transfer plasmids along with a plasmid encoding...

...want to: Generate complete and permanent loss of gene expression or function ( knockout )? Generate a specific... Edit Generate a specific user-defined sequence change in a particular gene, such as generating a point...like never before. This section will provide a general framework to get you started using CRISPR in your...Manipulation Application Cas9 gRNA Additional Considerations Knockout Permanently disrupt gene function ... enrich positive cells, or selection marker to generate stable cell lines (for details, see Plasmids 101...inserted into an AAV transfer vector and used to generate AAV particles (for details, see our AAV Guide ...Cas9 and gRNA In vitro transcription reactions generate mature Cas9 mRNA and gRNA, which are then delivered...

...curated literature information (Link opens in a new window) OMIM MECP2 - MECP2 curated literature information...MECP2 reactivation, RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to...contrast to the sustained loss of skills in neurodegenerative conditions, the regressive period in Rett ...locally or globally. The MECP2 protein contains several functional domains, including: the N - t erminal...acid and Thyroid hormone receptor (NCoR/SMRT) interacting domain (NID) a nuclear localization signal (not...the MECP2 protein in mouse and rat models have accelerated our understanding of Rett syndrome at the molecular...They are also an important tool for developing therapeutic strategies to treat Rett syndrome and restore...

...expressing firefly luciferase. pCMV-FLuc - Retroviral reporter vector expressing firefly luciferase. pHAGE-CMV-Luc2... vector expressing firefly luciferase and ZsGreen. NL4-3 mCherry Luciferase - Lentiviral dual reporter...EGFP. Lenti-luciferase-P2A-Neo - Lentiviral reporter vector expressing firefly luciferase with neomycin...poorly understood but are critical for developing therapeutic strategies to prevent viral spread. As a Biosafety...the live virus. Alternatively, it is possible to generate pseudovirus, where the structural and functional...spike protein is inefficient for pseudotyping; several modifications may enhance infectious particle production...lists expression plasmids that are suitable for generating pseudotyped virus for SARS-CoV-2 and other coronaviruses...

...the revolutionary dynamic picture of the brain generated through this effort will provide novel ways to...Hillel Adesnik 111066-AAV1 pAAV-hSyn-dLight1.1 To generate Adeno-Associated viruses for expression of dLight1.1...promoter Lin Tian 111067-AAV1 pAAV-CAG-dLight1.1 To generate Adeno-Associated viruses for expression of dLight1.1...promoter Lin Tian 111067-AAV5 pAAV-CAG-dLight1.1 To generate Adeno-Associated viruses for expression of dLight1.1...promoter Lin Tian 111068-AAV1 pAAV-hSyn-dLight1.2 To generate Adeno-Associated viruses for expression of dLight1.2...promoter Lin Tian 111068-AAV5 pAAV-hSyn-dLight1.2 To generate Adeno-Associated viruses for expression of dLight1.2...promoter Lin Tian 111069-AAV1 pAAV-CAG-dLight1.2 To generate Adeno-Associated viruses for expression of dLight1.1...

...systematic protein interaction screens or cellular assays to identify novel interactions or pathways that...method to detect interactions is AVEXIS (for Avidity-based extracellular interaction screen) in which ...protocol description: Avidity based Extracellular Interaction Screening (AVEXIS) for the scalable detection...of low affinity extracellular receptor-ligand interactions. Kerr SJ and Wright GJ. J. Vis. Exp. (61), e3881...Construction of a large extracellular protein interaction network and its resolution by spatiotemporal ...proteomics 2010 Dec; 9(12):2654-65. A cell surface interaction network of neural leucine-rich repeat receptors...for novel low-affinity extracellular protein interactions. Bushell KM, Söllner C, Schuster-Boeckler B,...

...EGFP Ken-Ichi Takemaru 54234 mEmerald-PLK1-N-16 Centrosome Plk1 mEmerald* Michael Davidson 54491 mCherry-Lifeact...Vladislav Verkhusha 79990 pKeratin-miRFP703 Intermediate Filaments (epithelial) Keratin miRFP703 Vladislav Verkhusha...mCherry Alpha Yap 54219 mEmerald-Paxillin-22 Focal Adhesions paxillin mEmerald* Michael Davidson 15233...GFP Bo Huang 54040 mEmerald-Clathrin-15 Clathrin-coated vesicles Clathrin mEmerald* Michael Davidson 79997...EEA1 GFP Silvia Corvera 42635 TagRFP-T-EEA1 Early endosomes EEA1 TagRFP-T Silvia Corvera 38770 pEF.myc....Optogenetics Fluorescent Protein Blog Posts Background General Cytoskeletal Junctions Secretory Pathway Background...structures as these well-characterized proteins. General Structures The cell contains a variety of organelles...

...highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide...Aug 13;11(1):16519. Takeharu Nagai Temperature Fluorescent temperature indicator B-gTEMP Intracellular Heat...You may also like... Addgene Blog: Biosensors Luciferase Plasmids Subcellular Localization Optogenetics...family Cameleon calcium sensors Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an... cAMP levels for imaging in live cells Fourth-generation epac-based FRET sensors for cAMP feature exceptional...GMP) Green fluorescent probe for cGMP (cGull) Generation of a cGMP Indicator with an Expanded Dynamic ...Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe. Nat...

...guidelines? A few notes below are considerations for designing HR donor. Generally if off-target (non-specific... PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but couldn't...In our hands, Herculase II Fusion polymerase or Kapa Hifi Polymerase work very well. Some groups have ...You may also like... Zhang Lab CRISPR plasmids General CRISPR Plasmids Dr. Le Cong, the first author of...concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately...additional PAM sequences should not be sufficient to generate off-target DSBs; there needs to be extensive homology...most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having a targeting...

...approach, stable lines with targeted genome alterations can be generated within a month. phsp70-Cas9 and a single...BbsI-crRNA : Generates crRNA for use in combination with tracrRNA. 51434 pHD-DsRed : vector for generating dsDNA...mediated genome modifications can be efficiently generated in Drosophila and transmitted through the germline...single chiRNA plasmid can be coinjected to generate mutations via imperfectly repaired DSBs. Coinjection...controlled by the regulatory sequences of an RNA polymerase III transcribed U6 snRNA. In both vectors, the...following template: example : Resources: Protocol for generating gRNAs 106.5 KB flyCRISPR website contains additional...96Ab promoter. 51019 pDsRed-attP : Vector for generating dsDNA donors for homology-directed repair to ...

...capable of controlling eukaryotic gene expression. Several endogenous promoters had been identified that responded... seven repeats of a 19 nucleotide tetracycline operator (tetO) sequence, and is recognized by the tetracycline...viewed in Krueger et al, 2004. Some cell culture sera may contain tetracycline or its derivatives which..., or TetR On or Off PI 21916 Tet-pLKO-neo 3rd generation lentiviral plasmid for inducible expression of...article for additional selection options third-generation rtTA On Fraser 44012 pInducer20 Lentiviral vector... article for additional tookit plasmids third-generation rtTA On Elledge 19407 pTREtight2 Promoter contains...single copy in mammalian cells; Expresses firefly luciferase hairpin and GFP under pTREtight promoter None...

...the new amino acid is introduced in the media. Generally, you will need two plasmids, as depicted in the...interest: The non-canonical amino acid, which is generally introduced in the media. A new codon to be allocated...in selected amino acids within a protein, any alterations in structure or function in the protein can be...and evolve proteins for various purposes from therapeutics to biopolymers. Tips for Success Before beginning...beginning to reprogram the genetic code, there are several things to consider. If you are working off of a...tRNA pair instead. For directed evolution of the liberated EcTrpRS/tRNA pair to incorporate other Uaas (derived...

...developing embryos. zfRegeneration - A database for gene expression profiling during regeneration. Seurat - R ...(embryogenesis is complete in ~3 days), short generation time (~3 months), ease of care and breeding, ...study of cancer, inflammation and infection, regeneration, human neurological disorders, microbiota studies...CRISPR Please refer to Addgene's CRISPR Guide for a general introduction to CRISPR technology or the zebrafish... Vivo Lineage Tracing during Development and Regeneration - Periklis Pantazis Lab Multibow: digital spectral...abundance, size, and localization in high-throughput. MAZERATI platform - Leonard Zon Lab. A platform to rapidly...Zebrafish Tol2 Gateway-compatible Kit for Neurodegenerative Disorders - Nicholas Cole Lab. A kit consisting...

...expression when cloning in E. coli . This alteration enables the generation of a single vector containing Cre ...separate DNA molecules, a translocation event is generated at the loxP sites. For a more thorough introduction...Drosophila. Read Addgene's Blog on Cre alternatives from Gerald Rubin's lab. Cre-containing Plasmids Scientists... Cre-EGFP fusion in a promoterless vector with several available restriction sites none Mammalian Sauer... region including the polyadenylation site and several introns tet-hCMV Mammalian Sauer 12168 pMB80 (R26...hsp70 Insect Rubin 32606 Ttr:Cre Cre expressed in visceral endoderm MCK Mammalian Hadjantonakis 33340 LGmCreER...backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR Cre, luciferase, and sgRNA expression EFS AAV Zhang 60229 AAV...

...estrogen receptor 1 DKFZp686N23123, ER, ESR, ESRA, Era, NR3A1 ESR2 estrogen receptor 2 (ER beta) ER-BETA...MGC3886 ERAP1 endoplasmic reticulum aminopeptidase 1 A-LAP, ALAP, APPILS, ARTS-1, ARTS1, ERAAP, ERAAP1, KIAA0525...disulfide isomerase family A, member 2 PDA2, PDI, PDIP, PDIR PDIA3 protein disulfide isomerase family A...KGFLP1 keratinocyte growth factor-like protein 1 MGC125746, MGC125747, MGC126891 KGFLP2 keratinocyte growth... TDGF1 teratocarcinoma-derived growth factor 1 CR, CRGF, CRIPTO, Cripto-1 TDGF3 teratocarcinoma-derived...systems. Upon infection, the immune system triggers several processes to activate cells to recognize and get...the dominant defense strategy during the first several days following exposure to a pathogen. The innate...

...suitable for generating SARS-CoV-2 spike pseudotyped virus. It also lists several luciferase and fluorescent...mammalian sequences linked to SARS-CoV-2 infection. General Tools : Plasmids expressing Cas12a/Cas13a, Reverse...top Plasmids Encoding Mammalian Genes or Inserts Several mammalian genes have been identified as having ... our Immunology Resource Guide . Return to top General Plasmid Tools SHERLOCK and DETECTR - Simple, portable...window) reverse transcriptase, protease, and Bst polymerase constructs now available from Andrea Pauli's ...nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2. Huo, et al. Nat Struct Mol Biol. 2020...Diagnostic Toolkit Enzymes. (unpublished) Taq polymerase and MMLV reverse transcriptase expression plasmids...

...an indel or inversion mutation [3]. In the CRISPR era, the genomic landscape may be surveyed in unprecedented...present a video reprint of the CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines ...between deletion size and frequency [3]. There are several applications in which the production of deletions...within the deletion boundaries may be produced to generate clonal allelic series. Single DSBs resolved by...

... a 5'LTR and should be packaged using the 2nd generation packaging system. Didier Trono Cre-regulated ...Description Tet on or off PI 11643 pLVCT-tTR-KRAB 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter...and Didier Trono 11779 pLVCT-rtTR-KRAB-2SM2 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter...Aebischer and Didier Trono 11644 pLVET-tTR-KRAB 2nd generation; Transgene (hEF-1alpha promoter) ‐ OR ‐ shRNA...Aebischer and Didier Trono 11642 pLVPT-tTR-KRAB 2nd generation; Transgene (hPGK promoter) ‐ OR ‐ shRNA (H1 promoter...and Didier Trono 11652 pLVPT-rtTR-KRAB-2SM2 2nd generation; Transgene (hPGK promoter) ‐ OR ‐ shRNA (H1 promoter...Aebischer and Didier Trono 11651 pLVUT-tTR-KRAB 2nd generation; Transgene (hUbiquitin promoter) ‐ OR ‐ shRNA...