...expressing oncogenes or toxins should be handled at BSL-2. AAV can transduce both dividing and non-dividing ...plasmid, expressing Rep2 and Cap1 Wilson 104963 pAAV2/2 AAV2 AAV packaging plasmid, expressing Rep2 and Cap2... are used in conjunction with the plasmid that encodes the viral genome (typically called cis -plasmid...plasmid). Helper Plasmid ID Plasmid Serotype Description PI 112867 pAdDeltaF6 All Helper plasmid for AAV...Packaging Service at Addgene ID Plasmid Capsid Description PI 112862 pAAV2/1 AAV1 AAV packaging plasmid,...

...the cells will still appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity...I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn’t ...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare, progressive...in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene activation...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.org/10.1038/13810... (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations. Am ...Tillotson R, Selfridge J, Koerner MV, Gadalla KKE, Guy J, De Sousa D, Hector RD, Cobb SR, Bird A (2017) Radically...that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides during neuronal maturation (Tillotson...

...postdoctoral researcher at Instituto de Investigación Biomédica de Salamanca (IBSAL) and one of the coordinators...mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results were promising,...receiving dozens of requests every day.    Figure 2: When a researcher needs a collaborator for a specific...Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert’s suitability and availability... illustrates the power of collaboration, which despite the competitiveness of cutting-edge research, is...physics. Even non-scientists, such as lawyers, designers and other professionals, joined the platform; ...

...Constitutive 1, 2, 5, 9 Deisseroth 26973 pAAV-hSyn-hChR2(H134R)-EYFP Syn ChR2/H134R EYFP Constitutive 1, 2, 5, 9....WPRE.SV40 CAG ChR2/H134R mCherry Constitutive 1, 2, 5, 9 Deisseroth 127090 pAAV-CAG-DIO-ChR2(H134R)-eYFP...ChrimsonR (soma-targeted) mRuby2 Cre dependent 9 Adesnik 124603 pAAV-EF1a-DIO-ChrimsonR-mRuby2-KV2.1-WPRE-SV40... ChroME (soma-targeted) mRuby3 Cre dependent 9 Adesnik 124651 pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 CaMKII...eGtACR1 (soma-targeted) mRuby3 Cre dependent 9 Adesnik 137155 pAAV-nEF-Con/Fon-iC++-EYFP nEF iC++ EYFP...

...thanks to our guest blogger, Harshana S De Silva Feelixge. Harshana S De Silva Feelixge is a researcher whose... post was contributed by guest blogger Harshana S De Silva Feelixge. Gene therapy technologies hold ...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates...form, translating gene therapies from conceptual design to clinical trials involve identifying a therapeutic...

...referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the...standard consisting of ECFP and EYFP connected by 2 GGSGGS repeats pET28CLY3 Peptide linker standard consisting...fluorescent protein. Plasmid Color Expression Description pPROEX Aqua Cyan Bacterial Expresses Aquamarine...

...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins...McLellan JS (2020) Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369:1501–1505....labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman... protein, variants, and pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the...of infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published...reagents for pseudotyping lentivirus with the SARS-CoV-2 spike protein. They deposited the plasmids used for...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts To make targeted epigenetic...catalytically dead dCas9 to epigenetic modifiers. Design your gRNA to target a specific promoter or enhancer...

...goals when designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8,...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression...as your titer changes, so may your needed volume (de Backer et al., 2010).  Second, high efficiency transduction... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),...–10. https://doi.org/10.1371/journal.pone.0106472 de Backer, M. W. A., Brans, M. A. D., Luijendijk, M.... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Catalytically dead dCas9...gene expression by interfering with transcription. Design your gRNA to target your gene of interest’s promoter...

...nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin ...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore...11;342(6155):253-257. PubMed. 3. Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda T, et al. Characterization...and Bauer lab's related paper describes their methods for CRISPR design, cloning, and delivery, as well...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Catalytically dead dCas9... can increase transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator...

...on opposite sides of the world shows that science is truly a global endeavor. Case study 2: Plasmids Another...repository provides. Repositories around the world facilitate the rapid pace of SARS-CoV-2 research by...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...Many of the reagents used to investigate SARS-CoV-2 were developed for broad use and are now important...susceptibility of cells from different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in...applications but have proven useful to the study of SARS-CoV-2 are the lentiviral production and packaging vectors...

...the use of pAdEasy-2 can increase the capacity of the adenoviral vector. pAdEasy-2 does not contain the...Adenovirus packaging plasmid that lacks E1 and E3 pAdEasy-2 Adenovirus packaging plasmid that lacks E1, E3, and...911E4 cells Cell line compatible with the pAdEasy-2 packaging plasmid; Supplies viral protein E4 E1 Supplied... Science Guides Adenoviral Guide Adenoviral Guide Components Vector...be harvested about 7-10 days later. Vogelstein designed multiple shuttle vectors for different purposes...

...suggestions, like alternative word suggestions. De-jargonizer The De-jargonizer can identify jargon in your writing...episode 2 "Organizing Literature" : I found out about Zotero here Dear Grad Student podcast episode 2 "Essential...This paper talks more about the development of the De-jargonizer.   Academic Phrasebank  The Academic Phrasebank... of the suggestions Hemmngway makes (it wasn’t designed for technical writing), it’s great at flagging... Just enter the DOI of a paper and select your desired formatting style. This tool is great for generating...library in Microsoft Word. Mendeley has a web and desktop version that works with Windows, Mac, and Linux...easier to work with collaborators. Zotero is a desktop based program that’s compatible with Mac, Windows...

...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts CRISPR/Cas nickase mutants...Selectable Marker PI Publication Yeast ID Plasmid Description Gene/Insert Promoter Selectable Marker PI Publication...

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... rather than aversive stimuli. In a recent paper, de Jong and colleagues used rAAV FLEx-based vectors ...brain regions associated with motivated behaviors (de Jong et al., 2019). FLEx vectors represent, therefore...developmental biology at the École Normale Supérieure de Lyon. Her love for educational science led her to... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...

...2007 ) in mammalian cells or zebrafish). pMG10 (a '“2-in-1” ZFN cassette for expression of both a “+” and...This page lists plasmid vectors designed to express nucleases in different cell types. Zinc finger arrays...Expression Vectors This page lists plasmid vectors designed to express nucleases in different cell types. ...zebrafish (can be used as a substitute for pMLM335 described in Foley et al., PLoS One 2009) pMLM292 (for expression...zebrafish (can be used as a substitute for pMLM336 described in Foley et al., PLoS One 2009) pMLM800 (for expression...Methods 2011 ) in mammalian cells or zebrafish as described by Moore et al. PLoS One 2012 and Cade et al.,...

....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...variants for widespread gene transfer to the adult brain. Nature biotechnology. 2016;34(2):204-209. PubMed...because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they have ... On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains ...stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction...cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...