...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...

... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites...the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the sgRNA sequence...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...

...Biotechnology, 10.1038/s41587-023-01756-1. https://doi.org/10.1038/s41587-023-01756-1. PMID: 37095348. Breaking... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and...expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under...StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587-023-02018-w. PMID...broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023-38582-7...StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery...

...successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This first ...and leave for 1 h at room temperature. After this, it is helpful to subject your membrane to several freeze... the membrane itself or to any “sticky” proteins expressed by your library. After blocking for 1 h at ...it can really help to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding...place to start) and add your membranes to the solution, shaking gently for 1 h at room temperature. This... on Twitter @JessicaSacher.   References 1. Nobrega, Franklin L., et al. "Revisiting phage therapy: new...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...

... PCR leads to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894...products are then transformed directly into chemically competent E. coli and transformants selected with ... for further purification before transformation. After transforming cells, you should perform colony ...consisting of digestion, purification, ligation, and transformation. While restriction cloning is still routinely...

...Davidson AR.  Nature. 2013 Jan; 493, 429–432. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...sequences were initially discovered in E. coli in 1987 (1), the concept that these clustered repeat sequences...neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the...and are not translatable to the other CRISPR subtypes. Still, the large and dynamic range of CRISPR systems...well-suited for various innovative biological and translative technologies. Addgene has empowered researchers...http://en.wikipedia.org/wiki/File:Crispr.png Barrangou R, Horvath P. Science. 315, 2007 Jan; 1709–1712...CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu...

...marker. Looking at Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart...called Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective ...efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining... the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores...Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores...blue laser excites at a wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation...Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation...

...of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using...on a range of topics including biotech and research tools, computing performance, and randomized control...

...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...fasting has a positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or ...of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn may represent...  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...discussed and Professor Caetano Reis e Sousa from the Francis Crick Institute concluded “Aspirin promotes autophagy... Kettering Cancer Center. In contrast to using randomly integrating viral vectors, CRISPR/Cas9 genome ...

...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.4] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...specific integrins in cellular processes.   Figure 1: Integrins can shape into three conformations: (A)...immunoprecipitation and immunohistochemistry. Table 1: IPI antibodies  Antibody Target Proteins Reactivity...Human IgG1 Rabbit Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG1 Rabbit Anti-Integrin...Human IgG2a Mouse Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG2a Mouse Anti-Integrin... β1 integrin mechanics. Nature Communications, 13(1), 7471. https://doi.org/10.1038/s41467-022-35173-w...

...between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group ...plating your transformation may allow you to see colonies within 8 hours. References: 1. Shuman S. "...Let’s break down the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers ...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...

...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the... and not in academia is run by grant funding. Learning to write grants quickly, efficiently and effectively...apply to or offer to help with lab grant writing. You may not get the grant or your PI may edit everything...thinking about future career options. 5. Write a grant - even if you don’t need one A lot of science in...valuable skill. There are many different kinds of grants that you can apply for. If you are in a lab, your...

...estimated to affect anywhere from 1 in 6,000 to 1 in 19,000 live births (1).  While disruption of the smn1... funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...disease. This year, Addgene will be providing plasmid grants worth $2,000 each to two labs through the BeHEARD...

...spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team will communicate...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you to choose...and are exceptions to our standard workflow.  Table 1: Guidelines for the data fields in the deposit spreadsheet... Send plasmid information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene...

...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

...near-perfect physical-to-infectious particle ratio of 1:1 (Zeltner et al., 2010). However, for recombinant ...preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene AAV...reported a physical-to-infectious particle ratio of 50:1 (Zeltner et al., 2010). The specific infectivity of... lot (if we still have it available). References 1. Fagone, Paolo, et al. "Systemic errors in quantitative... Human Gene Therapy, Part B: Methods 23.1 (2011): 1-7. PubMed PMID: 22428975. PubMed Central PMCID: PMC3640491...particles that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type....qPCR...Digital droplet PCR...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any ...

...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis... and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair. This method...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing...template plasmid, these PCR products are directly transformed into bacteria expressing recombineering proteins...fine, especially using plasmids in the 10-12 kb range. REPLACR-mutagenesis also requires making your own...

...type, and/or desired event.   Figure 1: Transduction to expression process. A viral particle enters...triple T’s of biology are transformations, transfections, and transductions! In this blog we will discuss...intricacies of transductions, we've got you covered there too. What is a transduction?  Transduction is the ... experiment. Using transduction vs. another method Transfection and transduction seem to achieve similar...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are amenable... process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar ...to find out more! Molecular basis of transduction Transductions utilize the ‘normal’ mechanism of viral...

...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...control and is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...

...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will...to additional protocols and plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "...