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  1. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...terminally differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...Time to expression AAVs have to infect cells, hijack host cell machinery, and synthesize a second strand...that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic, do not...be specifically targeted to a number of different cell types even when systemically administered in an ...ranging from transient gene expression in a particular cell type to CRISPR genome editing, optogenetics, and...

  2. Neurodegeneration Plasmid Collection

    Type
    Collection
    ...8661 p4455 FLAG-hPLIC-2 UBQLN2 Flag CMV ALS Peter Howley 8662 p4456 FLAG-hPLIC-2 NTF UBQLN2 Flag CMV ALS...196208 GB1-A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep...196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196211 GB1-A11(2-196, G175R...196212 GB1-A11(2-196, G189E)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ...TLS 1: hTLS.pBSKS(+) FUS T7 ALS David Ron 21828 TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C...ApoE Alzheimer's Sohail Tavazoie 51436 pGL3Basic-ME.2/ApoEpromoter APOE ApoE Alzheimer's Sohail Tavazoie...T7 ALS Nicolas Fawzi 127195 RP1B FUS 1-163 QQ4xSS #2 FUS His T7 ALS Nicolas Fawzi 127196 pJ411 FUS 1-163...

  3. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants...

  4. Rett Syndrome: A History of Research and Therapeutic Outlooks

    Type
    Blog Post
    ...//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...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 ...Syndrome and Related Disorders. Cell 152:984–996 . https://doi.org/10.1016/j.cell.2013.01.038 Guy J, Gan J, ...Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878 Sinnett SE...deliver the precise amount of protein to each brain cell to restore physiological levels. Much research is...

  5. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) ...rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...

  6. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression is... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... 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... where many different cell types, or even different phenotypes of the same cell type, are intermingled...postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells in far away...

  7. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    ... recombination at the cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki...2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit ...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...delivered using AAV injection, the majority of the cells surrounding the injection site will become infected... ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...used to study the physiology or behavior of certain cell populations.  Acknowledgement:Through a partnership...

  8. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

  9. Tips for Getting a Faculty Position

    Type
    Blog Post
    ... this problem? (2-3 sentences) What will be the focus of your first grant? (1-2 sentences) ...of scientific success is publications and grants. 2. Write succinct and easy to understand documents A...consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students (that are solicited...people would agree is important to understand. (1-2 sentences). For example: Distinguishing correctly ...patterning of cells and tissues is fundamental to development. I'm interested in how cells find their way...competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/corresponding... optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance...

  10. Hot Plasmids and Viral Preps - January 2021

    Type
    Blog Post
    ... Opto-Cre-Vvd is fast and can cut in as little as 2 hours.  Sheets, MB, et al., ACS Synth Biol. 2020....they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons...a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or...deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct and allows a ...fluorescence-activated cell sorting (FACS) helps researchers sift through thousands of cells to identify cells exhibiting...target single cells. Now, Francois St-Pierre’s lab developed a new form of FACS-based cell screening, Single-cell...individual cells with unique spatiotemporal phenotypes from large heterogeneous cultures.  HeLa cells expressed...

  11. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The genome-wide...target all protein-coding genes present in the genome; 2) non-targeting guides (red), which target no sequence...guides that had zero mismatch off-targets. Guides with 2-bp mismatch off-targets were only toxic if they had...tolerated than mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target...PMID: 28474669. Pubmed Central PMCID: PMC5424143. 2. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide...delivered as a pooled library via a lentiviral vector to cell lines that either stably expressed Cas9 or that ...almost all genes involved in the production of a cell surface glycan that’s required for ricin uptake....

  12. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...

  13. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...developing T-cell acute lymphoblastic leukemia due to insertional mutagenesis (Howe et al., 2008). 2. Transient...vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing... chromosome of the cell. This integrated viral genome is then part of the cellular DNA… forever!  This...viral entry into the host cell.  Transfecting these three plasmids into cells enables the production of...

  14. Viral Vectors 101: What is a Virus (and a Viral Vector)?

    Type
    Blog Post
    ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit...safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have been genetically...hijacking the machinery of the cells they infect. Once they enter a cell, they treat their viral genome...genome as a “payload” that is delivered to the cell. The cell then uses its own machinery to replicate the...replication process whereby one cell copies itself and “splits” into two cells. Viral vectors Viruses are ...virus. From one, come many After a virion infects a cell, the single particle creates many copies of itself...millions of new copies, which are released from the cell like baby spiders from an egg sac. (Okay, some enveloped...

  15. Technical Design of a Western Blot

    Type
    Blog Post
    .... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability ...separation of proteins of different sizes.  Table 2: Recommended gel percentages for various protein sizes...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...

  16. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ... and future challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332...Pluripotent Stem Cells, 2013, 23-33. PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke...Gene-delivery systems for iPS cell generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID:...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...

  17. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    ...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

  18. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ... used. Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...containing the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert...colonies than the vector + insert plate (unsuccessful 2), or roughly equivalent numbers of colonies on each... or equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid...use electro-competent cells instead of the more common chemically-competent cells. You should perform, ... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...

  19. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...Single-Domain Camelid Antibodies. Cell 181:1004–1015.e15 . https://doi.org/10.1016/j.cell.2020.04.031 Zimmermann ...coronaviruses and enable the virus to enter host cells. To do this, they injected the spike protein from...camelid antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  But llama ...

  20. Year of the Camelid: Antibody Style

    Type
    Blog Post
    ...and a source of sdAbs (Figure 2) (Xu, et al., 2021).      Figure 2: Genetically modified mice offer... from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature 595:278–282. https://doi.org/10.1038...antigens in living cells or tissues. sdAbs can also be used to manipulate cellular functions, like in ...sdAbs over conventional antibodies include:  Excellent tissue penetration, thanks to their small size...

  21. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine deaminase...editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular components...available for plant systems. Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful...based CBEs are known to deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer....PMID: 27096365. PubMed Central PMCID: PMC4873371.  2. Nishida, Keiji, et al. "Targeted nucleotide editing...homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery...Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an original C-G...

  22. Early Career Researcher Toolbox: Social Media for Scientists

    Type
    Blog Post
    ...scientists are using social media professionally, and 2) I needed to start using Twitter. Sharing Chemistry...but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn...PhD 👩🏾‍🔬💜 (@ThePurplePage) February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post...you social media can positively impact your career, 2) given you tips for using various social media platforms...Science Communication” at the American Society for Cell Biology (ASCB) 2018 Conference. I don’t exactly ...

  23. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome... genetic loci within a single cell. One might imagine transfecting cells with both the nuclease-positive...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as ...reliance on orthogonal Cas9 species. Within a single cell, full-length guides targeting one set of genetic...knockin mice for genome editing and cancer modeling." Cell 159.2 (2014): 440-455. PubMed PMID: 25263330. PubMed...

  24. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources... of cell types, so they’re ideal for work with primary cells or other difficult-to-transfect cells. Lentiviral...dividing cells. Another type of vector, based on adenovirus, could infect non-dividing cells, but without...ability to transduce both dividing and non-dividing cells. Lentiviral vectors are the vector of choice for...

  25. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

    Type
    Blog Post
    ...when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...- The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color variations...(prepared with Addgene #104110 - #104112) (Figure 2). We’ve also created constructs with containing chemical...imaging with single-cell resolution using chemical cocktails and computational analysis." Cell 157.3 (2014):... optimal expression of XFPs. Any longer and the cells may start to show morphological abnormalities due... wait too long, as too much XFP is toxic to the cells. Inadequate expression levels of XFPs - Reduce the...video below depicting dendrite wiring of mitral cells.   Many thanks to our guest bloggers Richi Sakaguchi...

  26. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

  27. Mouse Modeling, Part 2: Breeding and Crossing Mice

    Type
    Blog Post
    ...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...strain, the promoter being used should be tested in cell lines to determine how it impacts the expression...interest. Cre may be expressed globally (in every cell of the mouse) or in a tissue-specific manner (with...

  28. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids...precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV lysis buffer and freeze...bath to release the virus particles from the cells. Cell lysates are then treated with benzonase immediately...

  29. Viral Production at Addgene

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ... preparations are added to cells in culture. Three to five days later, cell cultures are inspected for...a new vial of early passage cells. Approximately two weeks post-thaw, cell culture supernatant is tested... (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445...preparations ("-LV” catalog items) are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging...

  30. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...imaging and loose-seal, cell-attached recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence...protocols for viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty...a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered to the cell surface by a GPI-anchor... Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA Puro ...either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control ...PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing ...particles in HEK293T cells with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260). HeLa cells were infected...

  31. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...expression in non-dividing cells because they remain in episomal form in the cell’s nucleus. But since nothing...hope are many more clinical successes for gene (and cell) therapies. One may wonder why the first AAV-based... providing long-term gene expression in retinal cells - AAV - were becoming available. At the same time...

  32. DNA Purification Without a Kit

    Type
    Blog Post
    ...level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image: Zou et al., 2018...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant...

  33. Adenovirus Guide

    Type
    Guide
    ...use of pAdEasy-2 (Addgene #16401) can increase the capacity of the rAdV vector. pAdEasy-2 does not contain...gene and cell-based therapies in the new era of personalized medicine . Genes & Diseases, 4 (2), 43–63.... incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with BioRender.com...and have to be handled at biosafety level two (BSL-2). They are highly immunogenic and can trigger a strong...adenoviruses using the AdEasy system . Nature Protocols, 2 (5), 1236–1247. https://doi.org/10.1038/nprot.2007.135...Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic . NPJ Vaccines, 6 (1), 97. https://doi.org...backbone plasmid that lacks E1 and E3. pAdEasy-2 Adenoviral backbone plasmid that lacks E1, E3, and E4. Useful...

  34. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...

  35. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...

  36. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

    Type
    Blog Post
    ...human transcription factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal... example through the use of less toxic adjuvants; 2) generating complex antibody chips that allow assessment...produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells are fused with activated...activated B cells from the spleen of mice injected with the antigen of interest. The immortalized cells, called...distributing over 3,000 antibodies and associated hybridoma cell lines. The DSHB was created by the National Institute... individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing...

  37. Optogenetics Guide

    Type
    Guide
    ...nonspecific cation influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...transcription effectors OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated... with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...

  38. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...Occupational Classification System  and Wikipedia. 2. Social and computer scientists are overrepresented...international Twitter users were included in the dataset; 2) users not part of a scientific Twitter list were ...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of scientist...tweeted by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific...

  39. IBEX Knowledge-Base: A Data Resource for Multiplex Tissue Imaging

    Type
    Blog Post
    ...imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038/s41596-021-00644-9 ...directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...

  40. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    ...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support open...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images...gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative...What works well in one cell or neuron type may not work well for a neighboring cell type, even within the...options while AAV8 (82%) and AAV9 (84%) were both excellent at infecting our target neurons (Figure 1). However... conditions (3,5,8), injection titers (1,2), and cell types used for packaging viral particles (6), can...

  41. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration into ...our website to search for your favorite gene! STEP 2: Generate an Expression Clone When making the expression...expression construct in only 1 day, as opposed to 2+ days with traditional restriction and ligation cloning...Clon Transgen 4:138. doi:10.4172/2168-9849.1000138 2. Hartley JL. Use of the Gateway System for Protein...the next step is to transform competent E. coli cells and select the positive clones. The entry clone ... expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few...ccdB gene. Since the ccdB product is toxic to the cell, Gateway cloning efficiency can reach >99%. ...

  42. The Importance of High Titer for AAV Transductions

    Type
    Blog Post
    ...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially...roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with an increase...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...Making contact with a cell Although an individual AAV particle’s ability to infect a cell is dependent on its...construction and the cell it’s infecting, getting these particles to infect cells is also very titer-dependent... particle to infect a cell, it has to come into physical contact with that cell. Since there is a limited...of surface area on each cell, the way to get more AAV in contact with the cell is to increase the AAV ...

  43. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure 2), IVA cloning can be...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch...transformation efficiency of the strain: when asking the cell to perform multiple recombination events, all linear...linear fragments need to get into the same cell. For simple cloning requiring just one or two recombination...recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications...

  44. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ...various components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of ...drug reaction. This system would somewhat act like a 2-step verification. All approaches have their own limitations...Future Enhancements. Frontiers in drug discovery, 2, 952326. https://doi.org/10.3389/fddsv.2022.952326Joubert...epitopes likely to be presented by T cells to elicit an immune response. T cell epitopes are found in either ...that take place within an antigen-presenting cell like a T cell. It is also not yet possible for these algorithms...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of the...CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or macrophages...

  45. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    ... were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer to distribution and...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...rapid adoption of new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates...As we celebrate twenty years at Addgene, we are sharing stories of our repository’s positive impact on... Sharing this story is a truly special way to celebrate our twentieth anniversary in 2024, and it’s all...

  46. University of Florida Serotype Testing Panel for the Eye and Brain

    Type
    Collection
    ...AAV2 vectors in the mouse retina. Mol Ther . Feb;19(2):293-301. PMID: 21045809 Other citations include: ...adipose tissue-derived mesenchymal stem cells into pluripotent stem cells by a mutant adeno-associated viral...serotype also demonstrates enhanced transduction of cells in culture for ex-vivo conditioning. The AAV2(trpYF...displays enhanced transduction of hematopoietic stem cells and microglia. The AAV6(dbY-F+T-V) serotype was ...AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates...human gene therapy of glaucoma and their reversed cell entry characteristics. Gene Ther . 2020 Apr;27(3...delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of...

  47. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...translocation. Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W.,...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...

  48. Antibodies 101: ChIP

    Type
    Blog Post
    ...everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP experiment! Image...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically... Process ChIP can be done for either tissues or cells. The process is very similar for both, though tissue...fragmenting the DNA, spin down the samples to remove cell debris, which will collect in a pellet. The supernatant..., these are mostly fairly standard molecular and cell biology techniques. What’s all the fuss? Well, ChIP...will also have to be optimized for both tissue or cell type, amount of protein, and desired fragment length...

  49. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ...Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell Tropism after Delivery to ...one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This ...flank the transgenes (non-homologous pairs) (Figure 2.). They found that almost all off-target expression...exercise caution when using them.    Figure 2. Schematic diagram showing the mechanism of spontaneous... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310...specific cell type rely on transgenic models that express recombinases like Cre or Flp in their cells of interest...entry into the cell (Dudek, AM. 2018).  If the serotype you are using transduces cells that you are not...

  50. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...particle sneaks its way into cells. Generally, AAV serotypes have a primary cellular receptor and sometimes ...transduction efficiency, and is an excellent choice for targeting cardiac cells (Bish et al., 2008). It can ...
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