...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some...either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response to Adenovirus...Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing a lack...adenoviral vector infected cells does not require de novo viral gene expression: Implications for gene...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature...viruses infecting humans (from seven species, designated as letters A–G) exist. The life cycle of AdVs...short-lived. It is worth noting that it is possible to design a vector that achieves persistent transgene expression...

...published an article on BioRxiv describing a comprehensive SARS-CoV-2 virus-human interaction network ...comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes an additional catalytically...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins in human cells and used these viral “bait...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D....changes that the biosensor is designed to detect resulting in false positives [2, 4]. Solutions to the polymer...an A206K mutation [2]. Addgene has many of these constructs and they are designated mGFP, mVenus, etc....analytical ultracentrifugation [2]. Some affinity assays used in the papers describing the FPs above including... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might...

...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...Strausbaugh LD, Gray P, Vartak NB, Brown M, Talbot D, Berg DE. pBR322-derived multicopy plasmids harboring large...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...

...-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org/10.1101/2020.04.16.045419.  Wrapp D, De Vlieger...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...Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly select for...strongly to your target protein. The Seeger lab designed 3 different mRNA sybody libraries for use in the... randomized with a defined set of different trinucleotides (corresponding to different amino acids) in...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...Solution: In Zhou et al, the Lin lab present a generic design for creating photoswitchable kinases and use MEK1...

...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally...bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated...on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes, as well... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...

...clear and detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification...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...392–400. https://doi.org/10.1038/s41434-018-0028-5 de Backer, M. W. A., Brans, M. A. D., Luijendijk, M....gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative...of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve our Addgene...every researcher experiences and that it often provides incredibly valuable insight into one’s work. As...

...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... Special thanks for Dr. Dan Stone and Harshana de Silva Feelixge at Fred Hutchinson Cancer Center for...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...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap) genes...replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged into the...

...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...to sickle cell disease. Hoban MD, Orkin SH, Bauer DE. Blood. 2016 Feb 18;127(7):839-48. PubMed PMID: 26758916... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources... but without sustained transgene expression. To design a viral vector system that could do both, Addgene...infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats...

...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ...SARS-CoV-2 virus. Scientists have been using similar pseudotyping methods with other viruses in a BSL-2 lab... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid...example, production of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl...for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12...Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116 Gutierrez-Guerrero...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the... site (Figure 2, top).  These primers are typically ~70 nucleotides long (20 nucleotides that anneal to... genomes. 2) Designing ssDNA oligos that avoid MMR activation:  There are two ways to design oligos that...that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E. coli with...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...

...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...media will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 ...in a 10-100-fold increase in virus. Purification (2 days) - Purification is required if rAdV is to be ...

...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...gRNAs. Photocleavable guides are generated by introduction of a single photocleavable 2-nitrobenzyl linker...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101: Ribonucleoprotein ...

...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...that are built into how something functions, were designed for this purpose. One such control is dividing...

... recombinase (Figure 2). Figure 2: A knockout targeting vector designed to insert and later remove...process, we’ll walk through an experiment designed to knock out exon 2 of a given gene.   Figure 1:...   Design your targeting construct. For recombination to occur in a cell, a minimum of 2 kb of sequence...multiple ways. The desired recombination event will remove NeoR only and leave exon 2 floxed, as seen in...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...often use the Cre/Lox system described earlier. In this case, you design your targeting vector such that...

...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...databases, DNA sequence manipulation, and primer design. Best wishes for the new year and your research...

...Rodrigo Arturo Rivera Solís, Universidad Autónoma de Yucatán, Mexico Dr. Rodrigo Arturo Rivera Solí... genetically encoded H2O2 probe is pQE-30-HyPer3 (2) which he will co-express together with a plasmid ...Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et al. “HyPer-3: A Genetically Encoded...renewable biotechnology products and biosensors for herbicides as well as developing yeast as source for dietary...technology to engineer plants that are able to destroy DNA viruses, similar to the anti-phage CRISPR effect...cloning kit) she intends to make yeast capable of desaturating fatty acids. Identification of Insecticidal ...as insecticidal proteins, the use of chemical pesticides may be avoided or decreased. Biosensors for herbicide...

...and 2), they found an indicator that is optimized for 2-photon imaging of voltages. This includes monitoring...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors show that...

...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...