...easily understood and consistent in design and layout. 2. Be concise in your writing  When writing a manuscript...worthless. But simply writing down your data doesn’t guarantee a publication. There is an art to composing manuscripts...

...affinity and specificity. Trends in Immunology, 29(2), 91–97. https://doi.org/10.1016/j.it.2007.11.004 ...kill the target cell. If the pathogen is a large parasite, ADCC can be induced through a very similar interaction...

...but also something about how science really works. 2)   Read science writing in magazines or newspapers...people like a good story and science involves characters – in this case you – on a quest to ask and answer...

...articles, such as: Sander JD & Joung JK, Nature Biotech, 2 March 2014. Or browse the articles related to the ...system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design but...

...PMID: 30356044. PubMed Central PMCID: PMC6200774.   2. Nielsen, Alec AK, and Christopher A. Voigt. "Deep...properties (GC content or rare codon percentage) and comparative properties (percent sequence identity and percent...

...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally... Kroeze et al. developed a new approach termed Parallel Receptor-ome Expression and Screening via Transcriptional...library of approved drugs against the entire kit; a parallel analysis that successfully identified new, highly... generated using slightly relaxed gRNA design parameters. To demonstrate the effectiveness of their library...

...acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...Cas12a’s ability to knock out multiple genes in parallel. Lastly, this Cas12a-based screening method was...gels. A: individually expressed proteins run in separate lanes. B: individually expressed and coexpressed...containing 150 or 250 kD proteins are also available separately. The proteins, which are purified via histidine...or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems in both gene activation...

...closely mimicking the future application. In figure 2 we provide an example of the comparison of the practical...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended...practical brightness takes all application specific parameters into account, including the specs of your microscope...translated from the same open-reading frame and separated from the protein of interest by a 2A self-cleaving...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive... was also between 1013 - 1014 vg/kg, a dosage comparable to those currently used in human gene therapy...response and brightness. They also do not spectrally separate from green sensors and blue-light-activated optogenetic...phytochrome-like fusion proteins from the plant Arabidopsis thaliana. In the presence of red light (650 nm... PCB is no longer present, the fusion proteins separate, inactivating Cre. This is critical for reducing...

...cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. “A glance at genome editing with...with a dominant mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of... explore its potential in mice with a dominant cataract disorder caused by a single copy of a gene known...

...responsible for that differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced...road map, and what was known about previously characterized TFs, the Zhang group were able to classify unknown...studied TFs, this functional association of the uncharacterized TFs is a useful finding. Transcriptional program...

...and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In...transformation to testing and a lack of well-characterized genetic tools make it challenging to engineer...understood. Additional work has also identified and characterized mechanisms and components of the circadian rhythm...

...gamma-retroviral vectors, you need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA, or...for different cell types (known as tropism). Figure 2: Gamma-retroviral plasmids. Created with BioRender.com...leukemia virus (FeLV). Retroviruses are partially characterized by their ability to integrate their genomes ... vectors, these required components have been separated into distinct plasmids for safety to reduce the...eliminating the need to deliver these genes on separate plasmids ( in trans ). This method, therefore,...The genes used for viral vector production are separated into three different plasmids containing the transfer... A retrovirus from the Retroviridae family. Characterized by long incubation and the eventual cause of...

...JW, Cammarata M, Perez M, Agarwal P, Brodbelt JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197...essential for the structure of paraspeckles. Clemson CM, Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess...

...IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the...isotype? An isotype determines several key characteristics of an antibody as well as the role it plays... regions result in several unique physical characteristics of these five isotypes, such as the number ...IgG4. IgE IgE antibodies help protect against parasites but also play a role in allergies. They are found...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...experimental approaches. One example is CasX, characterized by the Oakes and Doudna Labs, which is both ...research and potentially treatments. Cas12b, characterized by the Zhang (Strecker et al., 2019) and Li ... relative to Cas9. As scientists continue to characterize CRISPR nucleases from a variety of sources, ...

...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...from Pseudomonas aeruginosa to cleave 4 gRNAs separated by 28 base Csy4 recognition sites from a polycistronic...protoplasts. Each pair of gRNAs targeted genomic sites separated by 350-750 bp, allowing them to examine the frequency...different biological systems. Although Xie et. al characterized the system in plants, PTGs will likely work ...

...is the most charged, with 5 negatively charged and 2 positively charged amino acids. FLAG tag has been ...the need for an antibody. Tags can enable the separation of detection of a mutant protein vs. endogenous...

...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...micro- or nanofluidic channel and the sample is separated by electrophoresis. Smaller fragments migrate ...

...two options to begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a ...format. Figure 6: Add sequence, maps, and files. Step 2: Gene and Insert *Note: This page will not appear ... into it. You will have the option to indicate separate promoters, tags, or cloning information for each...

...156–159. https://doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method...gradients and ultracentrifuged for up to 20 hours to separate the intact RNA from DNA and proteins. Although...and Sacchi showed that RNA could be effectively separated from DNA and proteins by a simple extraction protocol...thiocyanate solution. However, instead of RNA separation using cesium-chloride gradients, water-saturated...ultracentrifugation!), the layers of phenol and chloroform separate, and RNA is retained in the top, aqueous layer...

...still depolarize neurons at lower MOIs. Figure 2: ChRgers are delivered by intravenous injection into... “designer” channelrhodopsins with desired characteristics. The model was trained to predict if a channelrhodopsin...these remaining sequences were selected for characterization with patch clamp electrophysiology. All thirty...named ChRger1, ChRger2, and ChRger3, were then characterized further by expression in cultured neurons and...performed well in a tissue culture dish, that doesn’t guarantee their success in vivo. Therefore, the Arnold and...

...main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying gene...need to be given some time to adapt to a testing apparatus. General stress levels of the test animal may ...the humans’ audible spectrum. The experimental apparatus should be cleaned thoroughly between animals as...beginning. Variability source Standardization parameters Animal Strain, age, sex, housing conditions...order to maximize possible effects. Injection Parameters: The injection needle should be as thin as possible...

..., but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple... other dNTPs. Once the vector and insert are (separately) digested to create 10–12 base “chewed back” ... T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete...

... and transfer 100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above...for processing several sequencing reactions in parallel. We have a few tricks to simplify the process....saves us the step of purifying the DNA and gives comparable success rates and sequence quality, which is ...

...retroviral library was used to transduce T helper type 2 (Th2) cells from mice that constitutively express ...to manipulate genetically. To solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR...

...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...10.3389/fmicb.2020.607028  Riley, R., et al., Comparative genomics of biotechnologically important yeasts...

...Without binding to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA...bacterial genomes consist of repeated elements separated by unique sequences. When researchers first discovered...

...to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling with subclass-switched recombinant...and validation pipeline. (B) Schematic shows the separate elements of the R-mAb expression plasmid involved...

...and in line with Addgene style.        Fig. 2: Reference image (left) provided to our graphic designer...the antibody guide always gets an expert review, separate from the author, and a second review from someone...

... adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively... wells with a purified protein allows you to characterize properties of an antibody that recognizes that.... As mentioned above, it can also be used to characterize antibodies against the antigen bound to the ...

...protocols 4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral...if preparing several different lentiviruses in parallel, this approach should be considered. Similar to...consider using a reverse transcriptase mutant in a separate control reaction. This will allow you to quantify...

... Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563. doi: 10.3390/ijms24021563 Resources on Addgene.org... therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a gene known...known as Crygc (Wu et al). They found that the cataracts phenotype could be rescued by zygote co-injection...polymorphisms (SNPs) in the TTR gene and is molecularly characterized by misfolding of the TTR protein, which contributes...

...products have distinct localizations. Figure 2: Split-wrmScarlet labeling proteins with different... than split-sfCherry3. It was created by Maria Ingaramo, Jerome Goudeau, Cynthia Kenyon, and colleagues...Cas9-Cas12a gRNAs under a single U6 promoter. The paralog and dual-targeting hybrid gRNA library can be used... used to identify genetic interactions between paralogs. The exon-deletion hybrid gRNA library deletes...

...more tips on using social media as a scientist here. 2) Public engagement - Participate in science festivals...book.  Many thanks to our guest blogger, Sarah Schmidt! Sarah Schmidt is engineering resistance against...This post was contributed by guest blogger Sarah Schmidt, a Marie Curie Fellow at The Sainsbury Laboratory...

... to remove this default setting. Timesaving Tip #2: blastx searches are inherently slower than blastn... BLAST button, consider the Program Selection parameter, as this will affect the amount of time to perform...BLAST button, you may have noticed the “Algorithm parameters” link. Click on this link to view advanced BLAST...have to remember to adjust any blastp algorithm parameters. We use the default scoring matrix BLOSUM62, ...

... those that also infect the liver.    Fig. 2: Summary of the generational evolution of the AAV ...SSAP system reports an efficiency of up to 20%, comparable to commonly used CRISPR/Cas9 methods for large...

...Era of Personalized Medicine. Genes Dis. 2017 Jun;4(2):43-63. DOI: 10.1016/j.gendis.2017.04.001 Maetzig.... 2011 Jun;3(6):677-713. DOI: 10.3390/v3060677 Varanda C, Félix MDR, Campos MD, Materatski P. An Overview...

...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...We will be covering these safety measures in a separate, upcoming post. If a viral vector seems like ...

... high-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware...in developing better, more sustainable, easily sharable products. Open-Source Microscopy Sharing Hardware...