... for gene disruption in Mycobacterium abscessus, an increasingly common human pathogen notorious for its... use in revealing antigenic differences between human, canine and simian isolates. J Gen Virol., 68 (Pt...luciferase is embedded in the I10 domain of the human titin protein and only exposed when the module unfolds...Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs.... E., Young, D. F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of monoclonal...39406745. Optimizing mScarlet tags for live imaging in C. elegans By Alyssa Neuhaus Red fluorescent proteins...

...sites (thrombin and TEV), and C-terminal tags (StrepII, 3xFLAG, 6xHIS, MBP, Myc). These constructs include...Spike Proteins. In: Methods in Molecular Biology. Humana Press, pp 331–338 https://doi.org/10.1007/978-1...

...80 °C. Titer All titering is performed on lentiviral preparations that have been stored at -80 °C and ...viral vector genome titers by droplet digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://...Infectious Titer by a Novel Droplet Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org...Preparations are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or ...preparations are then aliquoted, frozen, and stored at -80 °C. Concentrated lentiviral preparations (“-LVC” catalog..., and is routinely tested for mycoplasma contamination using mycoplasma detection kits. Cell line is maintained... tested for mycoplasma contamination. To date, Addgene has never had a case of mycoplasma contamination...

...difficult to grow. Growth Temp Choose from 30 °C, 37 °C, or room temperature. Growth Strain Please...Species of Gene or Insert Choose from: H. sapiens (human), M. musculus (mouse), R. norvegicus (rat), G. gallus...plasmid will produce anything dangerous or toxic to humans in bacteria, select “yes” in the drop-down and ...frog), D. rerio (zebrafish), D. melanogaster (fly), C. elegans (nematode), S. cerevisiae (budding yeast)... gentamicin, hygromycin, kanamycin, nourseothricin (clonNat), spectinomycin, streptomycin, tetracycline...maintain a glycerol stock. Choose from: ampicillin, apramycin, blasticidin, bleocin (zeocin), chloramphenicol...

...analyze the base editing capability of the seven human APOBEC3 proteins, finding that APOBEC3A and APOBEC3bctd...ACG start codon mutation, and integrated it into human and mouse cells that expressed either Cas9 or an...transient reporter for editing enrichment (TREE) in human cells. Nucleic Acids Research 47:e120–e120 . https...expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome would restore...codon that is converted to a ‘TAC’ or ‘TAT’ after a C-to-T base editing event. This edit changes the histidine... ATG codon from an ACG codon using a base-editor (C>T conversion) they could induce the translation of...Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464...

...02708-5 Hrit J, Goodrich L, Li C, et al. (2018) OGT binds a conserved C-terminal domain of TET1 to regulate...unusual patterns of DNA methylation detected in a human disease: cancer (Feinberg & Vogelstein 1983). The...1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301...89–92. https://doi.org/10.1038/301089a0. Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking...https://doi.org/10.7554/eLife.34870.  Kaas GA, Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR...lentiviral backbones expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck...

...target sequence downstream of the 7SK, human U6, mouse U6, or human H1 promoters. If you express fewer than...arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab...lab. pX333, a modification of pX330, contains humanized wtCas9 and two U6 promoters. To use this plasmid...destination vectors. Destination vector options include humanized wt Cas9, dCas9 (transcriptional repressor), and...plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional...used to create lentiviral vectors expressing wt humanized Cas9 and up to three gRNAs. Entry vectors containing...Tetsushi, et al. “Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system...

...the potential CRISPR technology can have on the human microbiome. You can consider the microbiome as a...and metabolic processes, occupying the gut. Many human diseases have connections to either specific organisms.... Engineering the bee gut microbiome Beyond the human gut, the Barrick lab has deposited plasmids that... of cytidine and adenine base editors that enable C-to-T and A-to-G base substitutions respectively. Substitutions...:723–728 . https://doi.org/10.1021/sb500351f Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG (2018... https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen LE, Sommer MOA, Nielsen AT (2016) CRMAGE...Vridhambal GS, Rivera AJ, Montgomery SA, Fortier L-C, Barrangou R, Theriot CM, Ousterout DG (2020) In Vivo...

...in vivo screening of human promoters, the Zhang Lab found a short fragment of human IBA1 promoter (hIBA1a...screens in human cells. Addgene offers vectors for CROPseq-multi using selection by Puromycin, Zeocin®,...various contexts in vitro and in vivo, including in human retinal organoids and live mice. Importantly, REVeRT...specificity of GFP expression for the indicated ssAAV5s. C) Quantifications showing high microglia transduction... microglia-targeting AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting...

...jcm.23.5.959-961.1986 Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., & Berkmen, M. (2012...not fully suppress Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi....References Hwang, J. M., Piccinini, T. E., Lammel, C. J., Hadley, W. K., & Brooks, G. F. (1986). Effect...Slow transformation   Spectinomycin/streptomycin Spectinomycin and streptomycin both inhibit protein synthesis.... Pros: Stable (spectinomycin). Cons: Cost (spectinomycin) or stability (streptomycin), doesn’t work for... for transformations. Con: Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits...Like carb, spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin...

...transversions C → TG → A A → GT → C C → G                   C → AG → C                   G → T A → C                   ...Rosser, S. J., Bi, C., & Zhang, X. (2021). Glycosylase base editors enable C-to-A and C-to-G base changes.... (2021). CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nature Biotechnology...deaminase could also perform cytidine (C) deamination, converting the C base to uracil (U). This is the same...given the pithier name CGBE1, for “C → G Base Editor 1,” and had a C → G editing efficiency of up to 68%...Beyond UNG: Optimizing C → G editors Now that several labs had shown that preferential C → G editing was possible...pyrimidines (C ↔ T).   Base transversion  Base changes from purine (A or G) to pyrimidine (C or T) or vice-versa...

... EA, Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated.../10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott DA... Yan M-Y, Li S-S, Ding X-Y, Guo X-P, Jin Q, Sun Y-C (2020) A CRISPR-Assisted Nonhomologous End-Joining...End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis. mBio 11: . https://doi.org/10.1128...

...calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume of virus ...of recombinant adeno-associated viral vectors." Human Gene Therapy ja (2019). PubMed PMID: 31140327. PubMed...viral vector genome titers by droplet digital PCR." Human gene therapy methods 25.2 (2013): 115-125. PubMed...Song, Neng, et al. "Detection of circulating Mycobacterium tuberculosis-specific DNA by droplet digital...

...There are over 40 trillion cells that make up the human body and G-baToN provides versibility in tracking...interactions New auxin inducible degron system for C. elegans New CRISPR plasmids New items from the viral...tool-kit for the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible... degron (AID) system has been extensively used in C. elegans since 2015 to conditionally degrade proteins...repression, and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity and influence...

...optimum of 30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular... neurons to form the functional networks of the human brain.     Acknowledgement:Through a partnership...identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance... receptor (ER) ligand binding domain (LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2 #14797). These...Sadowski. 1985. “The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently...

...(unless if you count normal human interaction for all of us studying human genes in test tubes and gels...Mello) These very cute birds learn their songs like humans learn speech. Finches must learn early, use auditory...existence, but most abundant cell on earth (twice the human biomass). The lab is developing a system using novel...habitats - even 50 meters from Old Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs...So, think beyond Drosophila melanogaster or Saccharomyces cerevisiae (both fine research organisms) -  ...

...production of lentiviral and retroviral vectors.” Human Gene Therapy Clinical Development 25.3 (2014). 175... for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided...divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a ... labs and hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to ...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...larger microbes such as yeast, fungi, or bacteria, mycoplasma can be extremely hard to detect with levels reaching...cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients and can...

...target a specific locus to create a knock-out in human embryonic stem cells.  Find GEmCherry2 reporter ...these methods a try.  References Højland Knudsen C, Ásgrímsdóttir ES, Rahimi K, Gill KP, Frandsen S, ...stably integrate a reporter gene that contains a puromycin resistance gene followed by the target site (the...genome edit:  A donor plasmid that contains a puromycin resistance gene linked to an in-frame H2B-GFP....

...3,300-fold in Drosophila cells and ~24,000-fold in human HeLa cells as compared to basal expression. Co-expression...also shown that the Q system is also functional in C. elegans. Subsequent work by the Luo and Potter labs...23995928; PMCID: PMC3811519.Seo SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene ...; PMCID: PMC5270762. Wendler, F., Park, S., Hill, C. et al. A LexAop > UAS > QUAS trimeric plasmid to ...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...He has also constructed a library of eGFP-tagged human ORF clones to allow testing and comparison of different... easy for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest...et al. "Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance....