...AdEasier®-1 cells (strain) - Bacterial strain that contains AdEasy®-1 plasmid...N-terminal Myc tag for mammalian expression pGEX-4T-1-3xMyc - Bacterial vector for Myc tag pETcon(-) - Yeast...PGKpuro2ABFP - For retroviral delivery of one sgRNA pMKO.1 GFP - Retroviral shRNA expression Retroviral backbones...expression under a CMV promoter pAdEasy®-1 - Recombine plasmids from the shuttle... Tet-inducible lentiviral shRNA expression pLKO.1 - TRC cloning vector - Lentiviral shRNA expression...driven puromycin Hygromycin Mammalian, Varies pLKO.1 hygro - Lentiviral shRNA expression lentiCRISPRv2 ...lentiSAMv2 - Lentiviral sgRNA cloning backbone pLKO.1-blast - 3rd gen lentiviral backbone for cloning and...

...Alpha-tubulin Microtubules 101786 ST6GAL1-mEGFP AICSDP-26 mEGFP Sialyltransferase 1 Golgi 109122 NPM1-mEGFP AICSDP...Institute ID Tag Protein Structure 87420 PXN-EGFP AICSDP-1 EGFP Paxillin Matrix Adhesions 87421 TUBA1B-mEGFP ...TJP1-mEGFP AICSDP-23 mEGFP Tight junction protein ZO-1 Tight junctions 91565 AAVS1-mEGFP AICSDP-35 mEGFP ...Centrioles 101782 LAMP1-mEGFP AICSDP-19 mEGFP LAMP-1 Lysosome 101783 MAP1LC3B-mEGFP AICSDP-25 mEGFP Autophagy-related... HIST1H2BJ-mEGFP AICSDP-52 mEGFP Histone H2B type 1-J Histones 109119 CTNNB1-mEGFP AICSDP-47 mEGFP Beta-catenin...

...Heidenreich 1, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F. Nat Biotechnol . 2015 Jan;33(1):102-6...SpCas9 alone without sgRNA. Full references are below. 1. SpCas9 (or SpCas9n, D10A nickase) + single guide ...screens. The libraries are available in two formats: 1 vector system - lentiCRISPR - sgRNA and SpCas9 together...):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed . Return to AAV In vivo plasmids AAV Plasmids...codon-optimized SaCas9 driven by either the ubiquitous cytomegalovirus (CMV, PX601 [#61591] ) or liver-specific tyroxine...

...Communications , 9 (1). PMID: 30082838 Maddalo, D., Manchado, E., Concepcion, C. P., Bonetti, C., Vidigal, J. A., ... most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism Engineered...from Staphylococcus aureus ) has a coding sequence ~1 kb shorter than SpCas9 while retaining the same basic...lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...efficiency and fidelity. Nature Communications , 13 (1), 1425. PMID: 35301321 Edraki, A., Mir, A., Ibraheim...interrogation by SpRY-Cas9. Nature Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein...CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa...

...Lentiviral BsmBI yes, cut S. pyogenes EGFP Ebert pLKO.1-puro U6 sgRNA BfuAI stuffer 50920 Mammalian/Lentiviral...Worm HindIII none S. pyogenes de Bono sgRNA with rpr-1 promoter 48961 Worm EcoRI none S. pyogenes de Bono...Zebrafish BseRI none S. pyogenes Zon pCRISPomyces-1 61736 Bacteria BbsI yes, cut S. pyogenes Apramycin...see plasmid page yes, cut S. pyogenes BFP Kuhn pLKO.1-puro U6 sgRNA BfuAI large stuffer 52628 Mammalian/...Puro Zoldos pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem(1/110) 71707 Mammalian hU6 yes, cut S. pyogenes Draetta...expression of Cas9, dCas9, or dCas9-VP64 along with 1-4 sgRNAs expressed from independent promoters. sgRNAs...for the expression of two gRNAs from Drosophila U6:1 and U6:3 promoters. Bullock pCFD5 Drosophila Utilizes...

... your account. We will notify you by email within 1 business day regarding your account verification. ... transfers to for-profit entities. The MTA is a legal agreement between the recipient and the depositing...authorized signatory is someone who can execute legal documents on behalf of the recipient institution...

...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars were less stable than ...and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation. Journal of virology 71:6174–6178...express these sugars. (Takeuchi et al., 1997). The alphaGal sugars end up in the envelope and are targets ...targets for complement-based killing by anti-alphaGal antibodies. The VSV-G envelope protein is inactivated ...

...using centrifugal columns. The final formulation buffer is phosphate-buffered saline containing 1 mM sodium...

...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778 Erdmann...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016... envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-40788-8 ... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3 ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central... system). The GAL80 repressor can bind to GAL4, partially inhibiting the binding of GAL4 to UAS. One can...domain (DBD) to GAL4 or VP16 activation domain results in chimeric GAL80-repressible and GAL80-insensitive...expression is low but is activated by GAL4 binding to UAS. If you place the GAL4 gene downstream of a tissue- ...

...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in ...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...genes from the pYES2.1 plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and... to grow in the absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation...

...  Request review: 1–2 days Prep and QC completed: 6–8 weeks Average shipping time: 1–2 days (USA); 3–4...choice of five different serotypes.   Figure 1: The process of requesting and ordering a Packaged...request in three different quantities: 10x 100 µL (1 mL), 25x 100 µL (2.5 mL), or 50x 100 µL (5 mL).  Once... production process. This process will take 1–2 days, and we anticipate approving the vast majority of...small subset of AAV plasmids will have technical or legal restrictions that make them ineligible for use in...submitted, the request will be reviewed by Addgene for legal approvals and technical compatibility with our production...

...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...museum "Fábrica Centro Ciência Viva" in Aveiro, Portugal. If cancer was a musician, then metastasize is...museum "Fábrica Centro Ciência Viva" in Aveiro, Portugal. She is particularly interested in science journalism...

...were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because the DREADD...plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural...This post was contributed by Adriana Galvan, an associate professor at Emory University School of Medicine...express hM4Di in neurons of rhesus monkeys or mice (Galvan et al., 2019). The hM4Di receptor is an engineered....     Thank you to our guest blogger! Adriana Galvan is an associate professor at Emory University School...2159–2165 . https://doi.org/10.1128/mcb.8.5.2159 Galvan A, Raper J, Hu X, Paré J, Bonaventura J, Richie...

...for the wild-type. However, variants such as the P.1 (Gamma variant) and the B.1.351 (Beta variant) were... tested at the time of this study. Figure 1: Neutralization levels against spike pseudovirus decreases...lowest levels of cross-neutralization occurs in the P.1 and B.1.351 variants tested. Image from Garcia-Beltran...References Cele S, Gazy I, Jackson L, Hwa S-H, Tegally H, Lustig G, Giandhari J, Pillay S, Wilkinson E...Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A (2021) Escape of SARS-CoV-2 501Y.V2 from neutralization...Feldman J, Pavlovic MN, Gregory DJ, Poznansky MC, Sigal A, Schmidt AG, Iafrate AJ, Naranbhai V, Balazs AB...

... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...he still reads “#” as a “pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation...experimental model organism. I then described the Gal4/UAS system used by geneticists to study gene function...favorite gene (YFG).  Sometimes, you want to use the Gal4/UAS system, but the available reagents do not match... or by filtering by fly as species of gene. Find Gal4 and UAS plasmids at Addgene Choosing a vector to...

...Weight Markers. Scientific Reports,7(1). doi:10.1038/s41598-017-02693-1. PubMed PMID: 28550309. PubMed Central...plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently deposited... PstI or EcoRV, these plasmids generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially... gels. Get Tips on Verifying Your Plasmid Table 1: Comparing Typical Comericial Ladders with the PSU...$10 to make 1000 lanes worth of the 100 bp or the 1 kb ladder. Using a commercial plasmid prep kit increases...Learn How to Analyze a Restriction Digest References 1. Henrici, R. C., Pecen, T. J., Johnston, J. L., & ...Additional Resources on the Addgene Blog Are you a Frugal Scientist? Save Money with the Baby Gel Box Find...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for... inducer levels. That is, if transcription factor 1 controls expression of transcription factor 2, which...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information...interest. These transcription factors consist of the GAL4 or ZIF268 DNA binding domains fused to either the...fluorescent reporters and other proteins controlled by GAL4 and ZIF268. To demonstrate independent control of... Aranda-Díaz et al transformed yeast with both a GAL4/estradiol controlled transcription factor and a ...

...supE44 thi-1 gyrA96 relA1 tonA JM109 Addgene; Promega e14-(McrA-) recA1 endA1 gyrA96 thi-1 hsdR17(rK- ...England BioLabs E. coli B F dcm ompT hsdS(rB mB) gal ccdB Survival Invitrogen F- mcrA Delta(mrr-hsdRMS-mcrBC...mB–) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20 (StrR ) xyl-5 λ– leu mtl-1 Top10 Invitrogen F- mcrA ...use, dilute your antibiotic into your LB medium at 1:1,000. For example, to make 100 mL of LB/ampicillin...Delta-lacX74 recA1 araDelta139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG tonA::Ptrc ccdA DB3.1 Invitrogen...(TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL...Delta-lacX74 recA1 araD139 Delta(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG Antibiotics commonly used...

...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles...