...established applications of the original hybridoma monoclonals, we anticipate that our chemically identical...kidney development. Despite their ubiquity and significance, these receptors have proved challenging to ...specific ligand-binding functions, all key to basic biomedical research and drug development. Integrin structure...long legs that protrude from the cell membrane, physically connecting the extracellular environment and ...signal cascades, and allowing agile responses to mechanical forces in the cell’s microenvironment. The actin...and (C) extended-open. When a ligand binds, it typically shifts the integrin from its closed to its open...resulting in cells that elongated and morphed asymmetrically (Jo et al., 2022). By contrast, LVDP-binding...

...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences... do need multiple first/corresponding author publications in quality journals. It helps to come from the...a job. The currency of scientific success is publications and grants. 2. Write succinct and easy to understand...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested...humbling. My conviction that I had a flair for communication was rapidly dispelled. She would ask me, "Is... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...

..., the lessons learned in pursuing one technological application can benefit many others and accelerate...vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs HIV-1 infects...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...create mutations in genes essential to HIV-1 replication. However, this approach could permit some viral...along with Cas9 in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed...-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number, although the ...these methods represent exciting advances in preclinical HIV-1 research that should be pursued in animal...

...would need to be much, much lower before clinical applications could be considered. It is important to ...answer questions of efficiency and applicability. Given moral and ethical concerns surrounding such research...experiments are needed before we can consider clinical applications for germline editing. Although this paper...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were... embryos carried indels at the mutation site, indicating that repair proceeded through NHEJ rather than...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure...consider that already-tested CRISPR alternatives are applicable in many situations. In the case of hypertrophic...

...previous two posts in this series described a practical approach to selecting a bright fluorescent protein...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity and has...most likely in an antiparallel configuration), typical structures known as Organized Smooth Endoplasmic...Constantini et al, 2012). The localization and biological properties of new fusions can be compared with...

...the 4D nucleome. Visualizing specific loci has historically been difficult, as techniques like fluorescent... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing... The technique successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated...distances correlating with the previously established physical map. In comparing pairs of targets ~2 Mbp apart...the respective hairpin binding domain. Pairs of identical hairpin sequences specify the primary colors blue... colors and a white composite. gRNAs with two identical hairpin binding domains produce blue, green, or...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live...

...simulate physiological gene expression regimes that cannot be easily achieved with classical gene overexpression...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly...light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors for... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...250 nm), and studying endocytosis is further complicated by the reliance on pH-based biosensors used to...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...SRpHi Biosensors here. Richardson et al. Nat Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 ...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...some reporters are better suited for particular applications. Read on to learn more about each reporter or...labeled tyramide molecule into a highly reactive radical, which is then covalently linked to residues within...however, compatible with most multi-labeling immunohistochemical protocols where either a directly labeled...

...science, dating back several hundred years, with historical ties to psychology and philosophy (the mind-body...there are two main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying...chemogenetic tools are designed channels that are chemically activated. Both are introduced in neurons and...excitation or inhibition upon light (or drug) application.  Genetic manipulation can also reduce expression...recombinase on transgenic flox animals to introduce modifications at the DNA level in a temporally and locally... brain area, avoiding side effects from such modification in other areas. An alternative strategy is to...in a certain type of behavior (for a review see Stoica et al., 2013). Vector delivery may also be achieved...

...in their ability to be specifically and potently activated by pharmacologically selective effector molecules...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...wiring of the brain and the underpinnings of neurological disease, neuroscientists need to be able to ...What is chemogenetics? Chemogenetics relies on genetically introducing engineered receptors that are only...et al., 2019). Using PSAMs PSAMs PSAMs, or pharmacologically selective actuator modules, were developed... such as varenicline, are already approved for clinical use. Varenicline has been further modified to ... create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download our chemogenetics...

...is complex Plasmid classification has shifted over the years, and new classifications are still being created... Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4)...us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome. Although...genes between bacteria can also have serious implications for public health. Bacteriocin plasmids: microbial...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred...which strains of E. coli contain this F plasmid is critical for successful Gateway cloning. In addition, scientists...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...remove the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et ...PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh...

...one be interested in state machine technology? Biological research has produced a massive amount of information...processes that are influenced by temporally ordered biochemical events. How do RSMs work? The state of an RSM...prescribed region of DNA termed the “register”. Each chemical input to an RSM drives expression of a distinct...performed on one register at a time. If a particular application makes it difficult to isolate single registers...state read-out method may not be suitable for applications that require minimal cellular perturbation. ...The input plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream...contains the register. Depending on the desired application, the inducible promoters we used to drive recombinase...

...relationships as part of the job application process can dramatically increase the chances of being seriously...demonstrate in your application). Beware the myths of scientist “overqualification” but it may not be ... of a resume can be crucial for a successful application. It takes time to prepare an excellent resume... Many job postings will result in hundreds of applicants the day they are posted.  If your resume is not... here for more on timely submission of a job application. 3) Read job descriptions carefully Be open minded...bold. You don’t have to have all of the listed qualifications to apply for a position.  A job description...wish list and the hiring manager knows that no applicant is likely to meet all the criteria. Note the criteria...

...Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. Stem...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ...differentiate only into myogenic precursor cells [5]. Classification of stem cells Stem cells are classified into...differentiate into cells of peripheral blood. Typically, there is a very small number of ASCs in each ...Limited. Only specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC ...of post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient...bypass the transplantation compatibility issues and ethical concerns of ESCs. As iPSCs can be sourced directly...

...must make is which system you will use, or more specifically, the type of cells that will be used to make...spanning both prokaryotic and eukaryotic organisms. Typically, what system is used is dictated by the specific...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do... one passage (transfection). This differs from typical insect cell preparations, which we talk about below... below. Processing The viral supernatant is typically collected over the course of a couple days to increase...supernatant will depend on the virus type. AAV is typically purified using methods like ultracentrifugation...

... many biological research situations, the relevant volume is that of the cell. For the physical behaviors...experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, ...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics...monomeric FPs are not actually monomeric, at least in practical terms. The propensity of a particular type of ... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...structures are large (microns), bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to...

...outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts ...

...strengthened my critical thinking and problem solving abilities, increased my technical know-how, and helped...the bench, look for “technical or product support specialist,” “field application scientist,” or “scientific...scientific or technical writer,” among others. Don’t discount job postings looking for PhD applicants since many... helped develop my science communication skills. Ultimately, my degree prepared me to be exactly the type... many will also accept applications from master’s candidates with experience.  Use LinkedIn to see what... you’re interested in (i.e. biotechnology, pharmaceuticals, etc.) Do their jobs sound interesting? Is ...deeper foundation of scientific knowledge and technical know-how that increased my autonomy and satisfaction...

...The fix/perm solutions can make a significant difference in the efficacy of your antibody. When compared...procedure enables quantification of antibody performance in major research applications. eLife, 12, RP91645...that the antibody being published doesn’t always indicate the presence of supportive data. To be supportive...that the antigen being detected in the intended application — here, flow cytometry — is the desired target... saponin. 400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb...multiple timepoints. When the target of interest is critical to cell survival or proliferation, an additional...this approach should also allow for the clear identification of the cell types of interest. By correlating...

...drives and their ramifications. Readers unfamiliar with academia’s challenges with publication and funding ...and CRISPR of course address some of the ethical implications of using these technologies, but also the...Connecticut College who has been teaching and communicating science for over two decades, attempts to answer... primarily on fluorescent proteins and their applications, tackles much broader issues in this brief volume...sexism in science, problems with peer-reviewed publications and funding, bioethics in the age of CRISPR,... and her essential-but-unlauded role in the identification of DNA’s double helix. Perhaps to the dismay...dismay of middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development...

...therapeutics. We’ll have more to say about clinical applications of CRISPR in the next section. In order ...and aceylation, respectively (these are chemical modifications, or functional groups, found on lysine)..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ... Table 1. Because of DNA’s 3D geometry and the chemical properties of modified histones, a genomic region...(ESCs). Your ChIP-seq finds just over 13,000 significant binding peaks for NANOG in the human genome. ...maintaining ESCs. So which of these 13,000 regions are critical? One hypothesis: the enhancers! This leads us ...approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have...

...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...bacterial host range. To further potential clinical applications, Ando et al. recently engineered synthetic...the bacterial population. But what if we could specifically target only virulent or antibiotic resistant...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such...antibiotics by their suppliers, and public health and medical organizations are working to help educate the public...encoded by a high-copy plasmid. In the case of the genomically encoded quinolone resistance, mediated by a single...represented here by cells of different colors, specifically targets bacteria with the given genomic or plasmid...

...networks in biological systems. NGS will also play significantly into the realm of clinical therapeutic...detection of KRAS mutations: diagnostic and clinical implications." The Journal of Molecular Diagnostics 12.4...validation process, also known as CRISPR genotyping, is critical to demonstrating causal relationships between ... Figure 1: Schematic representations of the indicated unbiased genotyping options. Figure reproduced...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...cell-based methods are genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by... amplicon sequencing (Hendel et al. 2015), a significant difference as CRISPR experiments move closer ...

... We’ve also created constructs with containing chemical tags such as the SNAP-tag, Halo-tag, and CLIP-...animals and fix the samples. When you use AAVs, it typically takes a few weeks to achieve optimal expression... Any longer and the cells may start to show morphological abnormalities due to the excessive amount of... levels of XFPs - Reduce the amount of tTA. Paradoxically, too much tTA leads to a reduced expression ...most likely by suppressing transcription. It is critical to express a minimal amount of tTA to achieve ...sensory systems focusing on the functional and anatomical maturation of the olfactory system.      Takeshi...University. His lab has been studying functional and anatomical aspects of sensory circuit development using ...

...basics but are here because you want to know the intricacies of transductions, we've got you covered there...The good news is that viruses are very valuable biological tools, and with the right safety considerations...infiltration into a cell. The twist is that the typical genetic cargo — the viral genome — is swapped, ...vector containing the cargo of interest is then typically transfected along with viral packaging plasmids...risk management when working with viral vectors. Typically, one day after transfection into the production...expression or integration resulting from transduction typically plateaus within a few days to a few weeks, depending...viral preps available for a variety of broadly applicable and popular viral vectors. Check out our viral...

...Effective verbal and written communication skills are key to publication and acquisition of funding. If...  Writing Communication Publicity Consulting internships Communication Teamwork Management...Models Communication Regulatory knowledge Networking Blogging Writing Communication Networking... skills such as leadership, management, and communication. A great approach to fully understand and acquire...find open positions and view their required qualifications and skill sets Taking the time to identify... science class: Science Club for Girls) Communication to diverse audiences Organization Mentorship...  Leadership Management Marketing Communication Business models Conference or club organizer...

...position at Addgene, the skeptical applicant asked if she would partake in “critical thinking” in the position... of time that doesn’t involve critical thinking. Former Addgenie Jessica Welch says, “[It] seems counter-intuitive...collaborating, managing, and communicating. After years of focusing on my technical skills with cloning and...involves a few key features that can be difficult to replicate in other work settings: The sense of being the...it. The opportunity to be creative and think critically. One of the very first times I interviewed a ...transferable skills. Transferable skills are the non-technical skills you pick up wherever you work, and you’...careers and desk jobs “alternative careers.” Realistically, based on the numbers of PhDs and available ...

...addition to certain staff scientists and those in technical roles. Therefore, institutions are responsible...suggested that the salary level should now be automatically adjusted every 3 years. In May 2016, Secretary...centrally administered, which leads to many complications, including the inability of some institutions...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...from $43,692 to $48,216, an increase of 10.4%. The Icahn School of Medicine at Mount Sinai in New York is...of the postdoctoral workforce. Based on our communications with and research on these institutions, we...to the new level:  We are still waiting for an indication of what (most of) Harvard, Johns Hopkins, (most...

...has and will continue to dramatically increase the number of CRISPR applications. The first non-Cas9 CRISPR... D1135E variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just as selective...Wolfe SA, Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome...doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo T, Park SW, Kim D, Kim...10.1038/ncomms14500 Find plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ...