... This allows them to replicate viral plasmids with the SV40 origin of replication. To generate a virus...proteins at high levels. In a case where clinical applications of recombinant proteins are necessary, HEKs...serum-free growth, which significantly reduces the cost of production. Research applications There are two areas...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent...cell biology, there are thankfully many other applications that they are useful for! What the HEK is it... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...

...Scientific Publication, where diverse approaches to peer review were discussed. Science communication opportunities...developmentally programmed regulation of DNA replication and gene transcription during vertebrate embryogenesis...with people, nurturing networks, as much as with technical knowledge. I leverage a breadth of knowledge to...I am constantly teaching – often explaining a technical piece of research to others outside that specialty...and jobs. At Science, which is published by the American Association for the Advancement of Science (AAAS...punctuation can be wildly funny as well as financially critical. Those who work on the publishing side of science...There is a rich and active community of science communication professionals who are thinking about, for example...

... also be perturbed by supraphysiological expression levels. BiFC quantification This is where BiFC outshines...Miller-Rhodes from the University of Rochester Medical Center.  You’ve probably heard of Forster Resonance...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...2018), the generation of such resources in less genetically tractable organisms lags behind. BiFC can also...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described...to the N- and C-terminal end of each PIP and empirically test which combination provides the best fluorescent...

...BglBricks: A flexible standard for biological part assembly. Journal of biological engineering. 2010;4(1):1. Pubmed... diverse functions, and thus, is used in many biological fields. In synthetic biology for example, high-throughput...elements to generate non-natural tools such as novel biological circuits or sensors. Given the expanding palette...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...transgene expression, or a variety of other clever applications. In one example, modular constructs generated...methods are suited for particular downstream applications. The tradeoffs between various cloning methods...

...biology. Therefore, my lab is dedicated to development of new biological technologies. The locus-specific...been working on development of technologies for biochemical analysis of genome functions such as transcription...technology, which uses locus-tagging and affinity purification, in 2009. However, iChIP requires insertion ... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic...describe this technology and some of its potential applications? Fujii: We generated a series of retroviral ...researchers to use enChIP in a wide variety of biological systems. We are currently developing several ...interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences. Thus, we've made ...

...to directly activate and inactivate biological processes. Chemical effectors are suboptimal for a number...neuroscience. In fact, precise light-based control of biological processes is highly useful in other fields, including...consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength can... also be used to engineer new pathways with sophisticated gene expression controls. Most cell types do...system to develop a bacterial camera that prints a chemical image, as well as a genetic method for the computational...light; these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive...; only two plasmids are required per system. Applications of these systems The large dynamic range of ...

...Luciola italica)Southern Russian firefly(Luciola mingrelica) 562 64 Luciferin ATP No Jamaican click beetle...Luciferases are a class of enzymes capable of catalyzing chemical reactions in living organisms resulting in the...gene, such as luciferase, usually serves as an indicator of transcription within cells, where detection... is measured resulting from the catalysis of a chemical reaction requiring luciferin, ATP and oxygen as...Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity laser...Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon...Background Lower Higher Post-translational Modification Not Required Required Photobleaching/Phototoxicity...

...antibody interaction and/or the downstream application. Typically, if the target protein will be detected...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Immunoprecipitation Overview An immunoprecipitation reaction is typically carried out in one of two ways. Both methods utilize.... 2) An antibody immobilized on a bead binds specifically to the blue triangle protein but not the other...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this... or magnetic beads with commercially available chemical agents. Directly conjugating the primary antibody...interactions To prepare for an IP, samples are typically lysed in a non-denaturing buffer containing non-ionic...

...fields where communication and writing are key skills. Other fields, often highly technical or skills-based...proactively address questions about your application. These could be logistical, like briefly explaining a gap ...excellent communication skills” try giving an example, like “I have extensive communication experience...usually yes! A good cover letter won’t hurt your application, but it can certainly help you stand out.  In...consider cover letters an important part of the application. Even within an organization, there can be variance...good cover letter!) As with any part of the job application process, it's a good idea to talk to people in...work on [phenomenon X], I read several of your publications on [specific topic]. I am extremely interested...

...revolves around synthetic humans called replicants which are genetically engineered by a powerful corporation...fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the prototypical base editor...genetic code of living organisms and its societal implications even before the discovery of genes themselves...explores the concepts of animal testing and modifications. The premise of the novel involves Dr. Moreau...technology led to the development of many important medical and agricultural products. But it also made SciFi...2018) were highly successful in the box office, indicating the insatiable appetite of the moviegoers for...a theme park.  Released in 1997, GATTACA is an American SciFi film starring Ethan Hawke, Uma Thurman and...

...and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then...Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting was performed...transduced with Cas9 expressing lentivirus at the indicated multiplicity of infection (MOI). Anti-vinculin...lines generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and...need to use FACS (like they did in the original publication). I thought I could see GFP expression with a...

...prior publications to go on, a submitted manuscript could only have been viewed as a significant improvement...causes malaria). I attribute their success to technical expertise, thoughtful single guide RNA (sgRNA)...the piñata builders - the reviewers. After some physical exertion on the part of the party goers, the piñata... solutions that facilitate most experimental applications in CRISPR engineering. When compared to that...easy to fault the lack of features in a single application. The question is then, should we use all tools...those engaging in CRISPR design, but it is a significant hurdle made worse by an ever increasing marketplace...the other hand some software tools make use of empirically derived and/or prior knowledge to inform on the...

...natural redundancy of biological pathways;  to observe a phenotype, the modification of multiple genes is...recognition and cleavage. This sequence flexibility indicates that gRNAs could be incorporated into these constructs...the 5’ end. PTG construct schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage...) How many loci can Cas9 modify? To test the application of this system to gRNA multiplexing, Xie et al...deletion with 4-20% frequency at the four loci, indicating that Cas9 can be guided to at least 8 distinct...multiplexing further illustrates the potential of PTGs. Applications of PTGs The major advantage of the PTG system.... Though the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely...

...Stanford University. Sex and gender are critical components of biological research that are often forgotten ...provide a table indicating the sex of the most commonly used cell lines appearing in American Journal of Physiology-Cell...the past decade. For more practical help, there are numerous methodological articles to consider—we have... that of human clinical trials. And, as Janine Clayton points out, late-stage clinical studies that draw...effects of using all female cells and tissues, specifically in stem cell therapies. Let’s go back to why... disease being treated, and other hormonal, immunological, and environmental factors. How to include sex... (NIH) now requires that sex be included as a biological variable in all NIH-funded research. Janine Clayton...

...normalization and loading controls needed for relative quantification of a western blot — and why you might want ...accurately compare different lanes? In relative quantification, the POI bands are normalized to the loading...other errors in experimental design and gel quantification. Figure 3: In a perfect world, protein...On this gel, the protein of interest band is significantly lighter in “condition 1” than “condition 2.”...and it’s also problematic for computational quantification. Outside the linear detection range, band intensity...common error associated with Western blotting quantification” (Gilda & Gomes, 2013). To address this source... Gilda & Gomes, 2013. Figure 7: Semi-quantification of liver lysate using different loading controls...

...proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation patterns...Depending on the research question and downstream application, one or several of those techniques can be used...populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...cell membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane...intracellular labeling with antibodies requires chemically pre-treating the cells to allow for entry of ...but preserving the cellular state (think of mummification). Permeabilization, as the name suggests, perforates...

...current CRISPR research focuses on its potential applications for human medicine (Waltz, 2016), the potential...engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a ...commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species including ...engineering using CRISPR, highlight the specific modifications to CRISPR machinery that allow for the use of...CRISPR principles). However, plant-specific modifications to commonly used CRISPR plasmids are necessary...selected the correct CRISPR components for your application, it is time to deliver these components to your...detergent, Polyethylene Glycol (PEG), although the application of this approach is limited to protoplast cells...

...exclude duplicate events, and the Live/Dead gate to focus your analysis on what’s biologically active....exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably..., you’ll first need to learn a little bit of theoretical background on the different parameters that your...and W describe the shape of a histogram, which graphically represents the time and intensity of the cell... fully depend on your individual experiment. Practical example: lymphocytes in a mouse tumor sample You... You made it! So much about the theoretical background. Let’s dive into the praxis of gating strategies...show up in a straight line one the plot, while duplicates deviate from that pattern. A.                                                                                                         ...

...strategies Publication System nickname Photoactivatable moiety Cas9 variatn CRISPR protein application Split...improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...enzymatic activity. Photoactivatable genome modifications by NHEJ and HDR using split systems Later in...mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency of 7.2%). The authors additionally...C714, generating paCas9-2. This change did not significantly alter the system’s efficiency at generating ...particular interest since blue light has limited applications in vivo. UV and blue light is readily absorbed...

... do anything. Viruses are complicated and they perform complex biological tasks, both of which are factors...C virus RNA quantification as measured with branched-DNA technology." Journal of clinical microbiology...factors in the technical challenges they can present. Many researchers find themselves plagued by troubleshooting...hail Marys that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors...vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying...Different titering methods can reveal differences in physical versus functional titer. If you have low titers...cells, replace the medium with half the volume typically used (e.g., use 5 mL medium on a 10 cm plate)....