...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...human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor ...

...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...introduce a desired genetic alteration. Listen to Our Podcast Interview with Alex Chavez Figure 1: Schematic...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The gRNA conventionally... truncated such that it exhibits 15 or fewer nucleotides of complementarity to a target site, Cas9’s DNA...truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated to result in decreased...

...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...

...been characterized. See Box 1 in Kudo et al. for more details on the design of the bNLS and NES sequences...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find the KTR plasmids here...reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with...fluorophores can be multiplexed together. Challenging  to Design: FRET reporter activity depends on both the distance...advantages. First, they are easy to multiplex. Their design allows flexible swapping of fluorescent proteins...overlap are used together. Second, KTRs are easy to design. The key determinants of KTR function are the positions...

...range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the dilutions, they...note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies (GC) per reaction. Since...In the image to the right, there is approximately 1 positive copy per microliter in the no template control...elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation...diagnosis." Emerging microbes & infections 7.1 (2018): 1-9. PubMed PMID: 29691363. PubMed Central PMCID: PMC5915492... to a new plate containing the mastermix which includes primers and a ddPCR supermix. Note that the supermix...

...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Pooran's NgAgo Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the...the cell, the same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene ...Try NgAgo for Yourself On the other hand, the widespread success of CRISPR-Cas9 across multiple model ...

...modifications). Ready to get started?   Figure 1: Break free from the ball and chain!   Epigenetics...?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to...doi.org/10.7554/eLife.34870.  Kaas GA, Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR, Song H,...Recently, however, a particular tool has gained widespread popularity – and notoriety – as a tool of choice...leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest. Having..., too), but if this is not possible due to gRNA design restrictions, anything within 5 base pairs should...

...Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene expression." Cell 159.4...detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined... health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid, Low-Cost Detection ...Specifically this includes primer directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9...is an Assistant professor at the Biodesign Center for Molecular Design and Biomimetics and the School of...development of diagnostics. Mostly transmitted by Aedes mosquitoes (aegypti and albopictus) and, aided by... toehold switches, which allow for the rational design of RNA sensors that can detect virtually any RNA...

...undergraduate students (1, 2), but educators face significant challenges in designing suitable projects. 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...Over Time project The Pathways over Time project provides a framework for these undergraduate research questions...

...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation...three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon ... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...modification allowed the deletion of release factor 1, the protein responsible for terminating translation...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...site-specific incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...

...very helpful to confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize...two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used...primers that bind at either side of the undesired portion, designed to amplify the rest of the vector sequence...

...kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... produced Ethical concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation...

... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...limits of oligonucleotides. These donors traditionally have larger homology arms in the 500 bp to 1 kb range...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...knock-in. Then choose which Cas enzyme to use and design a gRNA to where you would like to introduce the...sequence to introduce, which is done through the design and use of a donor DNA molecule. Your donor molecule...

...Figure 1 for a summary of the experimental results. GeoCas9 RNP editing in mammalian cells Besides being...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...from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while...

...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...

...  Figure 1: Using Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA...the target sequence is present in the pool of nucleotides, the nonspecific RNAse activity of Cas13a becomes...

...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...that are built into how something functions, were designed for this purpose. One such control is dividing...

...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought... a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette. Expression of Cre in...in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral ...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...Over the past decades, neuroscientists have made great strides to map brain regions and allocate specific...for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...

... Figure 1: In conventional gene targeting (A), you must first generate your desired mutation in mouse...much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR... drug induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid...CRISPR ribonucleoproteins.” Genome Biol. 18 (2017) (1):92. DOI: 10.1186/s13059-017-1220-4 PubMed PMID: 28511701... using long ssDNA donors.” Nat Protoc. 13 (2018) (1):195-215. DOI: 10.1038/nprot.2017.153 PubMed PMID:...engineering using CRISPR nucleases. Genome Biol. 19 (2018) (1):25. DOI: 10.1186/s13059-018-1400-x PubMed PMID: 29482575...using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To design a knock-in allele at a given...

...Fig. 1). So, while organic solvents may improve antibody binding for some targets, they can destroy it ...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...critical to the outcome of your IF experiment and deserve careful thought and attention.   * There are other...