... funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777372....proteomic interactions represent the framework that provides biological context. Assays monitoring impedance...sub-cloning into a wide range of Gateway-compatible pDEST vectors. ID Plasmid Gene/Insert Industry PI Pooled...solute carrier family of proteins. Library ID PI Description Human SLC Activation Library 132561 Superti-Furga...

...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...

...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...

...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...

...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...

...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...

... : An encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast and lentiGuide-Puro...Off-Spotter: tool for CRISPR/Cas design Program for designing optimal gRNAs. Provides feedback on number of potential...the Chan Zuckerberg Biohub . gRNA Design Deep learning models to design gRNAs and predict their efficiency... a new window) : a webtool that provides models for SpCas9 gRNA design. DeepBE (Link opens in a new window...window) : a webtool that provides models for base editor (BE) gRNA design. DeepPrime (Link opens in a ...window) : a webtool that provides models for prime editor (PE) pegRNA design. PMID 37119812 (Link opens...

...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...Therapy, 17(2), 150–157. https://doi.org/10.1038/gt.2009.135. PubMed PMID: 19847206 Desfarges, S., & Ciuffi...due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you ...Brugman, M. H., Pike-Overzet, K., Chatters, S. J., de Ridder, D., Gilmour, K. C., Adams, S., Thornhill,...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...been a staple in molecular biology for over three decades. Widely used across various research applications...and human papillomavirus (HPV), to name a few (Desfarges & Ciuffi, 2012). One family of viruses, retroviridae...

...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...

...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

...none S. pyogenes de Bono sgRNA with rpr-1 promoter 48961 Worm EcoRI none S. pyogenes de Bono pMB60 47941...is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme activity, experimental...BclI-SwaI none S. pyogenes URA3 Wyrick pCRISPomyces-2 61737 Bacteria BbsI yes, cut S. pyogenes Apramycin... S. pyogenes Zhang gRNA Cloning Vector Bbs I ver. 2 85586 Mammalian BbsI None S. pyogenes H Fujii pX601...pyogenes LEU2 Lu pDestTol2CG2-U6:gRNA 63156 Zebrafish BseRI none S. pyogenes Zon pDestTol2pA2-U6:gRNA 63157...module vectors, including three designed for dicots and three designed for monocots. Using these gRNA ...Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts CRISPR requires that you...

...Cre-ERT2 CAG Mammalian Heller 112622 pVHC_PGKneoLox2DTA.2 Venus, Cre-ERT2, targeting vector with MCS for homology...homology arms Mammalian Heller 112623 pEHC_PGKneoLox2DTA.2 Emerald, Cre-ERT2, targeting vector with MCS for homology... arms Mammalian Heller 112624 pmTHC_PGKneoLox2DTA.2 TFP, Cre-ERT2, targeting vector with MCS for homology...arms Mammalian Heller 112625 ptdTHC_PGKneoLox2DTA.2 tdTomato, Cre-ERT2, targeting vector with MCS for ...25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed . Ventura A, Meissner A, Jaenisch R, Jacks...efficiency, selecting desired clones, and making transgenic mice. ID Plasmid/Article Description Expression System...Cre expression plasmids. Addgene's viral service provides high-quality and high-titer viral preparations...

...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...

...dCas9VP64 and sgRNA from separate promoters. Table 2. pmax dCas9 Activator Expression Plasmids ID Plasmid...Table 4. Gateway Destination Vector ID Plasmid 48222 pAC90-pmax-DEST : Gateway Destination vector derived...the dCas9 protein provides the transactivation activity, the sgRNA component provides the specificity and...and can be transferred to Gateway destination vectors (e.g., pmax-DEST [48222] and (Link opens in a new...also like... CRISPR Guide CRISPR Protocols gRNA Design Tools CRISPR Blog Posts CRISPR-on is an RNA-guided... and is designed to target promoters or enhancers of genes of interest. Different flavors of activators...new window) Lifetechnologies Gateway Destination Vectors ) to adapt to different systems by LR clonase ...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

...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 ...

...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 Two classes of base editors...protein (Mu- GAM). While many base editors are designed to work in a very narrow window proximal to the...

...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

...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...