... an envelope are called enveloped viruses (Figure 1). Like bacteria, only a tiny fraction of viruses are...are pathogenic (Balloux & van Dorp, 2017). Figure 1: A non-enveloped virus (left) and an enveloped virus...create far safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have... https://doi.org/10.1016/B978-0-12-800947-5.00004-1 Taylor, M. W. (2014). What Is a Virus? In M. W. Taylor...reverse transcribe their gRNA into DNA, which is then transcribed into mRNA before being translated into ...genome types that can be either double stranded (ds) or single stranded (ss) (ie, dsDNA, ssDNA, dsRNA, positive... of virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case...

...Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed PMID: 26702451. PubMed Central PMCID... 2005 [1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac...integration [8].   Figure 1: Mechanism of the piggyBac™  transposase/transposon gene modification system...limit [1]. The piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap...The predicted frequency of ‘TTAA’ in the genome is 1 in every 256 base-pairs of DNA sequence, making it...technologies and strategies for their projects. References 1. L. E. Woodard and M. H. Wilson, “piggyBac-ing models...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...

...including yourself as the researcher.   Figure 1: Separation of retroviral packaging components onto...these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that...lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus...required whenever there’s a splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time...isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may...looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate... plasmid). Viral production then proceeds by transfection of all three plasmids into a producer cell line...

...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site will...genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of...infected and would have the potential to express the transduced reporter gene. Generating a FLEx switch to control...

...buffer at temperatures ranging from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...). G. stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature...or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome...Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and...

...HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central PMCID: PMC6610143...fused to a fluorescent protein. “Frankenbody,” like the infamous frankenstein, was constructed by grafting...called this probe the HA frankenbody (Zhao et al., 2019).  To develop HA frankenbody, the Stasevich lab tested...can you do with the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound...also proved that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA...acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ability to...properly once inside cells.  Developing the HA frankenbody To combat the issues with these antibody based...

...tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope... 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al., 2005)     References and ... on different viral membranes, tropism, and advantages and disadvantages: Table 1 (Joglekar et al., 2017... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...virus to either restrict or broaden the host cell range (tropism). Pseudotyping is only done on enveloped...plasmid, and (3) the packaging plasmid. These are transfected into target cells which produce lentiviral particles...

...holds true in genome-wide screens.   Figure 1: Summary of the advantages of the new approach to ... in both screens, with the growth screen having a 1% false discovery rate and identifying >88% of previously...in Morgens et al. contains three types of guides: 1) targeting guides (blue), which target all protein-coding...off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets had greater toxicity than ...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated than.... When comparing the toxicity of guides that have 1-bp mismatch off-target sites, short guides had reduced...Knockout Library are available from Addgene! References 1. Morgens, David W., et al. "Genome-scale measurement...

...infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous recombination...sequences of interest to cells with efficiencies of 1-40% after G418 selection (Kohli et al 2004, Kim et...with polylinkers for the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor...since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there are ...efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%. While this is comparable...gene inactivation in human cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ...

...). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...serotypes 1, 8 and 9 share conserved mechanisms for anterograde and retrograde axonal transport. Hum Gene.... (2009). Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat ... Brain Res 406(1-2), 317-321. PubMed PMID: 2436717. Towne, C., Schneider, B.L., Kieran, D., Redmond, D.E.Jr...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...

...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges in ...6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway with ...sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/V5-His...selected from different parts of the Met pathway (Fig. 1). In addition to the complementation experiments that...about Clare at the Biology Dept. site.  References 1. American Association for the Advancement of Science...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ...

...842 . https://doi.org/10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, ...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous...

...the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below) ...complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA duplex. Image from...allowed sequence replacement rates of approximately 1%. This is by no means a large number, but it was attained...who worked on this publication:         References 1. Richardson, Christopher D., et al. "Enhancing homology-directed...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...

...methods to improve AAV production.     References 1.Clark, K. Reed. "Recent advances in recombinant adeno-associated... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high...be required. For example, retrograde AAV gets transported up the cell’s axon. While the mechanism for this...yet fully understood, we can imagine that this transport is probably not 100% efficient. So the few AAV... higher retrograde AAV titers for successful transduction. In another example, neuronal gene expression..., fewer channel proteins are required for the transduced cell to become light responsive than fluorescent...process of generating viral particles. Limit tube transfer - the fewer opportunities your viral particles...

...vector-exosomes), exosome AAVs, or exo-AAVs (Figure 1). The researchers discovered that these particles ... are often found with cellular exosomes, small membrane vesicles or microvesicles (approx. 40 - 150 nm... diameter) that are secreted from the cellular membrane into the culture medium. AAV particles are either... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...to modify it to improve yields. For example, transfecting the producer cell line with an expression vector...proteins, nucleic acids) which possibly improve transduction properties. György and colleagues found that...that the association of AAV particles with membrane vesicles protects them from antibody recognition and...

...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs). Small edits require as little as 30...Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.  Additional Resources on the Addgene...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...

... microscopy contrast technique in modern biology (1). When performed correctly, fluorescence microscopy...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...of modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...

...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico...material is right for their experiment.     Table 1: Most popular guides, protocols, and collections of...in 2023, from visitors in 218 countries. In Table 1, you can see our most popular educational resources... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid... package stays in transit for a certain number of days and tracking average transit time for each courier...

...minutes, which was fun to watch. A simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at...to direct cleavage the result was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the...this type of experimentation to the eventual translation to the clinic and cures. There is no doubt that...

...Issue 19, 1 October 2000, Pages 3752–3761, https://doi.org/10.1093/nar/28.19.3752Wong M; Medrano J. Real-time...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...in primer Yes Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from...show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak, indicating...changes in ΔΔCT are represented by RQs between 0 and 1. Flipping your experimental and control values during...see this fold change as a positive difference (RQ>1), which may be easier to conceptualize. Just remember... in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_3, © Springer Science+Business Media New...