...Arabidopsis (CIBN), which they call the LADL Anchor. Using specifically designed gRNAs, they can then ...Inducible Degradation (AID) uses plant hormones called auxins to conditionally and reversibly degrade ... for AID. By tagging proteins with a 7kD degron called mAID and expressing the essential plant protein...microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered Marine, Anion-conducting and Intensely...publicly available metagenomic datasets with biophysical studies and the potential of mining datasets ...transcription factors tethered to dCas9, require long time scales with constitutively expressed proteins that are...nature of genomic looping due to it’s briefer time scale and its inducible and reversible nature. Find the...

...different signals in a certain region or calculate a colocalization factor to assess the spatial relationship...microscope to see where the target protein is (localization) and how much there is (quantification). But...protein targets and you want to know if they co-localize at the same place in the cell? Or you know that...inactivate the fluorophores in the sample, either by chemical treatment or photobleaching or by stripping out...yourself stuck with red-green images generated automatically from the microscope software, you can always...it, the secondary antibody may be binding non-specifically – try optimizing your blocking and washing conditions... your multiplex-labeled sample to see if the localization and intensity patterns are similar between the...

...Castano Corsino, a junior faculty member at Harvard Medical School/Brigham and Women’s Hospital (BWH). Based...like IMP, bring closer than ever (although not physically) the ideas and experience from talented professionals... IMP USA-Europe not only provide the specific technical and logistic support to increase the chances of...professional opportunities that are outside of our typical boundaries, and will arrive to Spain at one point...international mentors. Universities from five different geographical locations in Spain were enrolled: the University...represented a diverse group of individuals with technical and career path needs, and came from the universities...this first program. Although not everyone got a physical award, all the students experienced great improvements...

...began with only a few inventory items offered domestically, but by the end of 2016, we expanded our viral...2014). It is a dual-vector system, meaning it is typically used in conjunction with a second vector that ...lentiGuide-Puro. The alternative single-vector systems typically express Cas9 and a gRNA from the same plasmid....several benefits. First, the low viral titers typically associated with Cas9-expressing lentiviruses will...Tips for using this plasmid Cas9 expression is typically cytotoxic, which makes generation of high titer...particular signaling pathways in specific cell types (typically neurons). While all of these viruses are popular...expressed in cells that express cre recombinase. Typically, this feature is used to attain cell-specific ...

...Table 2: Effect of Localization on Rosella’s fluorescence emission. Localization pH Emission Color ...improves upon them. Methods for studying autophagy Biochemical assays Long-lived protein degradation: This... Pho8Δ60 mutant protein is expressed. Pho8Δ60 localizes to the cytosol in an inactive form unless autophagy...’s slow and only measures bulk autophagy. Morphological assays Electron microscopy: Electron microscopy... tagging it with GFP allows for tracking the localization or accumulation of pre-autophagosomal structures...will emit red fluorescence regardless of its localization in the cell. SEP’s pH sensitivity means Rosella...color fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and...

... mechanisms. Most take advantage of chemical groups intrinsically present on antibodies, such as the primary...Chemiluminescence, which is light produced by a chemical reaction, can be detected using specialized imaging...reactions produce a color change, so they are typically used in microscopy-based assays like immunohistochemistry...conjugates (especially if you start to consider clinical applications). But there are a few other classes...somehow be tightly linked to your antibody. This chemical coupling can be achieved through several different... made to antibodies, such as inclusion of non-canonical amino acids or peptide tags. 5) Strong protein-protein... productive lives without thinking about these chemical reactions. After all, there is an abundance of...

...hM4Di was fused to mCherry. To investigate the localization of the hM4Di, we used primary antibodies against...allow a high-spatial resolution of the protein localization. Surprisingly our observations at the electron...expression of the transgene of interest and its localization in the membrane. The lesson we learned from ...Electron microscope images showing ultrastructural localization of hM4Di in monkeys. The pre-embedding immungold...arrows point, respectively, to gold particles localized in the cytoplasm or bound to the plasma membrane...However, in some cases the fluorescent signal may be critical for the experiment. Since the fused protein could...Research Center. Using a combination of in vivo and anatomical techniques, her research aims to gain a deeper...

...through our lab using barcoded tubes and plates. The critical pieces of information for a given plasmid are ...plasmid sequence. Other information may also be critical to the maintenance and use of certain plasmids...for the process. Always sequence verify Once the physical sample and its associated information are at Addgene... but, in most cases at Addgene, we check what we call the “insert,” (the gene or genetic element cloned...recover around 750 bp of clean sequence from a typical reaction, or about 250 amino acids worth, so make...comparable success rates and sequence quality, which is critical for the efficient handling of large numbers of...glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then select forward...

...Research at universities typically requires funding from a variety of government and academic institutions...to trusted students. For example, if the proposal call aligns with your work, your advisor may want to ...actively pursue. Proposal writing: Improves technical writing and communication skills, Elucidates...required for a successful grant application. It typically includes overview information, key dates, funding...proposal and deliverables to the aims requested in the call. Step 2: Develop a research plan Most grant applications...delivers strength to the sentence. Below are the typical sections in a NIH RFA, for example, and tips to...met in order to deem your proposal successful. Typical proposals consist of 3 to 5 aims, with secondary...

...many have called out antibodies as major culprits in the reproducibility crisis of biomedical research ...why validation is so important. Antibodies are a critical component of the researcher toolkit due to their...other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets already exist...-out approach to show that an antibody binds specifically to the expected protein. Common approaches include...that you want to use to look at the subcellular localization of a protein of interest. Conveniently, you ...plasmid not encoding this protein, which we will call Negative Control Gene (NCG) (A). Then you lyse the...experiment and want to validate that your antibody is specifically capturing your RNA-binding protein of interest...

...most Enterobacteriaceae. An update to this system, called pEcCas/pEcgRNA, allows it to be used in more E.... CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable Elements by...-Assisted Targeting) and the Zhang lab's system called CAST (CRISPR-associated Transposase) both enable...eukaryotes (Qi et al., 2013). As in other systems, catalytically dead (dCas9) targeted to a promoter or gene ...gene body can repress transcription by physically blocking the elongation complex from binding the DNA or...responsive promoter to drive expression of a catalytically dead Cas9 enzyme allows reversible repression...Bikard et al., 2013). By targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to ...

...classrooms, the laboratory experience is focused on classical embryology techniques such as basic observation... They also created an online companion resource called “cutting class” which provides more detailed materials...technique that detects the temporal and spatial localization of mRNA transcripts in tissue. This is useful... manipulation. The technique involves using synthetically made RNA (riboprobes) with modified nucleotides... added to the tissue. The last step involves a chemical reaction to stain the cells that contain this ...easily generate off of a circular plasmid via PCR. Typically, to make these constructs, developmental biologists...target mRNA and silences its expression. More specifically, during the process of RNAi, an enzyme dubbed...

...Almost all patients have achieved persistent immunological reconstitution with a normally functioning T...translating gene therapies from conceptual design to clinical trials involve identifying a therapeutic gene,...carcinogenic profiles have limited their usability in clinical settings. On the other hand, vectors derived from...Unlike most naturally occurring viruses used in clinical trials, wild type AAV is not associated with any...http://www.abedia.com/wiley/vectors.php). These clinical trials have been directed at diseases including...HIV. A notable landmark study showed significant clinical efficacy against a form of blindness known as ...rAAV2 carrying the human gene RPE65, patients with clinical diagnosis of LCA and mutations in RPE65 (n=3),...

...they perform complex biological tasks, both of which are factors in the technical challenges they can ...hail Marys that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors...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)....increase transduction efficiency. These reagents typically enhance the adsorption of a virus particle to ...measured with branched-DNA technology." Journal of clinical microbiology 37.6 (1999): 1683-1686. PubMed PMID... virus adsorption on target cell membranes." Biophysical chemistry 97.2 (2002): 159-172. PubMed PMID: ...

...cable is threaded through—and fixed to—an optical insulator called a ferrule (below). The fiber optic cable...maintained during the entire protocol, otherwise optical stimulation will be erratic, opsin activation/inactivation...stimulation parameters must also be determined empirically but it’s best to try those that have already ...flexibility in behavioral experiments. However, the technical demands of using these wireless devices are significantly... in cerebellar Purkinje cells. The Journal of biological chemistry. 2011;286(29):25848-58. doi: 10.1074... Sparta DR, et al. Construction of implantable optical fibers for long-term optogenetic manipulation of...PMC3466483. 4. Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics...

...Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. Stem...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...directly from a patient’s adult cells when used therapeutically, the chances of post-transplantation rejection...Kushi) Mukherjee is the Editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. She...

...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... 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...produce baculovirus, a family of viruses that specifically infect insects. This is what makes insect cells...transfer plasmid and the baculovirus construct, called a bacmid. Like AAV and lentiviral packaging plasmids...

...smaller libraries, chemical transformations can also be used. Efficiency for chemical transformations is...expected transformation efficiency, or more specifically calculated based on the size of and weight of your...that of electroporation, but an advantage is that chemical transformations are generally cheaper and easier...easier. One library that Addgene distributes with a chemical transformation amplification protocol is the Chong... KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated Isomers (iSPI) Libraries ~11,000...DOCX, 17 KB)   For CRISPR libraries, Genome-scale CRISPR-Cas9 knockout and transcriptional activation...

...to your normal control - you can set up your calculations so that your assay output number (RQ value) ... In that case, an RQ value of one (or, more realistically, between 0.80-1.20) would mean you have one ... on and so forth. This also serves as a light technical control - an RQ value of 1.5 means you have either...incredible discovery. The range of RQ values used to call copy numbers should be as small as reasonably possible...all double-stranded DNA, while Taqman uses an intercalating dye, with a fluorescent probe and quencher to...). Sybr, therefore, cannot be used to accurately call copy numbers, though it has been shown to identify...all researchers. Accuracy needed Do you need to call exact copy numbers or just general trends? How confident...

...graduate student culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We ...public. My experiences in grad school, though often called “nontraditional,” helped prepare me to start CINSO...educating the public and organizing events. I contacted local professional organizations and nonprofits. I volunteered...nontraditional” career path long ago. Use your analytical mind to test and discover what career suits you...mistake, I evaluated what lessons I had learned – call it “troubleshooting.” Trying out new experiments...followers. It’s also been a great place to reach out to local establishments such as museums, professional organizations...State Museum and others when I was trying to find local informal science educators, and ever since, the ...

...MCC develops and makes freely available technical and pedagogical resources, offers mentoring for instructors...best teachers and mentors pass on more than just technical knowledge, shaping their trainees’ very way of...their research capabilities and laboratory and critical thinking skills (Knutson et al., 2010a,b). Classroom-based...at other institutions to form a support network called the MDH CUREs Community (MCC) that seeks to reduce...student enthusiasm, and research backs up that view (Callahan et al., 2022).     Fig. 2: Students in...Chapter 17, Einstein Discovers America. New York./  Callahan, K., Peterson, C., et al. 2022. External collaboration...classroom-based undergraduate research experiences spanning chemical biology, biochemistry, and neurobiology. CBE Life...

...Produce Chemically and Functionally Distinct Vectors. Molecular Therapy - Methods and Clinical Development...and community concerns AAV vectors are complex biological reagents; many factors, like storage buffers ...out of fear it points to flaws or gaps in their technical ability. But, especially for AAV data, that may...experimental settings. It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER...Hub. These minimum data standards are designed specifically to allow scientists to evaluate how small changes...evaluate AAV vector performance. Yet, most of the technical data needed to replicate and build on others’ ...word of mouth” as their source for this type of technical know-how, which highlights the dearth of AAV vector...

...Balancing research with medical practice as an MD/PhD. Darrel does 20% clinical work 80% research and was...for Regenerative Medicine at Boston University Medical School. Darrell’s research focuses on the lungs...Center for Regenerative Medicine, and within the biomedical research community. Along these lines, Darrell...Pathology and Laboratory Medicine. He attends in the Medical Intensive Care Unit, the Pulmonary Consultation...the Pulmonary outpatient clinic, all at Boston Medical Center. In addition he conducts basic stem cell...Regenerative Medicine of Boston University and Boston Medical Center. Special thanks to our sound engineer/producer...

...often scarce and difficult to identify. So what specifically should you do? What follows are just a few suggestions... in fact, some of the very things that were so critical to me landing my own position! Conferences One...Society for Cell Biology (ASCB) or the American Chemical Society (ACS) are just 2 examples of organizations...will sometimes result in a recruiter giving you a call. Many companies enlist the assistance of recruiters...appropriate job candidates. If you do receive such a call, make sure to get that person’s contact information... get the specific position he or she originally called about, you can keep in touch and reach back out...know all in one place! This information can be critical to a focused and effective job search. As with...

... Predoctoral Fellow at University of Rochester Medical Center. During development, complex genetic programs...central nervous system (CNS). To address this technological gap, a trio of recent papers describe the development...Cre under neuron-specific promoters, including calmodulin-dependent kinase II alpha (aCaMKII; excitatory...with different untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional...demonstrated by the Qi Lab, which developed a subgenome-scale CRISPRa screening approach in embryonic stem (ES...Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently a Ruth... Predoctoral Fellow at University of Rochester Medical Center. He studies gene-function relationships ...

...Within that research group, you will also typically receive technical mentorship from a ‘bench mentor’. In ...receive after you’ve found your mentor, though. Typically, you will identify a ‘scientific mentor’: someone...scientific project. Your scientific mentor is typically a professor who operates a research group. Within...component. Documenting during training It’s hard to recall a lot of details, especially when you are in a ...to do? Walk through the protocol mentally (or physically where relevant) and identify any gaps or questions...bring them to your mentor. Pro tip! Consider physically walking through the protocol, by going to each...experiments. Unlike in lab courses, where everything is magically made up by helpful TAs or assistants, and sitting...

...have been used since the 1930’s in a range of biological studies, including investigations into environmental... are powerful resources for investigating any biological process. High quality genome sequence A high-... mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish...thousands of mutations in genes affecting diverse biological processes—not just development—are available ... zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes, morpholinos...One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the...Naturally, the best model organism depends on the biological question, and zebrafish are not ideal for every...

... the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC)...have tended to avoid cell culture in favour of biochemical assays followed by in vivo testing. The mantra...Estimates of the amount of money wasted in basic oncological research due to contamination are in the tens...prevented and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach Scientist...experience through working for a variety of pharmaceutical companies including GSK, AstraZeneca-MedImmune...talks and seminars on cell culture and other pharmaceutical topics. The LGC Group also provides standards...Learn about the Dangers of Using GFP for Protein Localization Resources on Addgene.org Find Plasmids for...

...for neuronal/CNS expression. Modulated by calcium and calmodulin. GAL1, 10 General expression mRNA Yeast... promoters only work in prokaryotic cells and typically only in the same or closely related species from... be ways to turn them on or off at will with a chemical, heat, or light. In the cell, promoters themselves...scientists have created synthetic promoters, which typically include some combination of other promoter elements...Inducible with Tetracyline or its derivatives.  Typically contains a minimal promoter with low basal activity...CaMKIIa Gene expression for optogenetics mRNA  Ca2+/calmodulin-dependent protein kinase II promoter Specific...phosphage dehydrogenase Constitutive  Very strong, also called TDH3 or GAPDH. ADH1 General expression mRNA Yeast...

... request for phages to the directory does not typically come out until nearly the last minute when all...all other options have been exhausted. Theoretically, phage alerts can be sent out to the directory earlier...team must decide which labs should receive the clinical sample to test their phages. Because the sample...phages targeting organisms closely related to the clinical sample. For treatment, it is best to identify ... only used phage in basic research but not therapeutically: not all labs in Phage Directory have the resources...resources or know what it takes to prepare phages for clinical use. There are also different regulations from... individual phages. Integrating lab protocols, clinical information, phage research job postings, and ...

...Chinese Academy of Medical Sciences University of Texas at Austin University of California, San Francisco  ...scientific community support open sharing. But, one critical part was missing when the COVID-19 plasmids first...Children's Hospital                           California Institute of Technology             Johns Hopkins...                           National Institute for Physiological Sciences Broad Institute                                       ...part of research projects, another way is to systematically offer thousands of constructs for tagged and...enables research on solute carriers (SLCs) for medical research Recently, the Michael Davidson Fluorescent...

...also more precise. Base editors, for example, typically edit all the target bases within the base editing... target nucleotides are positioned within the canonical base editing window, base editing has higher efficiency...products to be indels while the efficiency of HDR is typically less than 10%. In contrast, the original paper...maximum efficiency isn’t everything — prime editing typically needs to be optimized for each application, and...nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which had prime editing efficiencies...optimized for human codons; additional nuclear localization sequences; and two mutations in Cas9 previously... 3′ end, the Liu lab appended RNA pseudoknots, calling this longer RNA an “engineered pegRNA” or epegRNA...

...GFP is tagged with a nuclear localization sequence, allowing it to localize to a different area of the ...mOrange2, EGFP, and mKate2 (red) to solve the problematically low fluorescent intensity of previous versions.... XFPs are introduced using a plasmid construct called Zebrabow-GateDest containing an RFP, CFP, and YFP...developed a version of Tetbow that incorporates chemical tags instead of XFP to optimize it for tissue ...architecture of a fluorescent neuron is visible. Chemical tags are more resistant to tissue clearing methods...neuronal circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed PMID: 30454553...homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells." Cell 143.1 (2010)...

...short-lived biotin-phenoxyl radical in the presence of hydrogen peroxide. This radical reacts with electron-...capture these interactions in a vast array of biological contexts.  Proximity labelling relies on the ...and determine the topology of the mitochondrial calcium uniporter (Martell et al., 2012). It is important...RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein interaction detection...Cell–Cell Interactions." Journal of the American Chemical Society 141.5 (2019): 1833-1837. PubMed PMID: ...et al. "Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy." Nature...total number of protein molecules per cell volume? A call to rethink some published values." Bioessays 35.12...

... important proof of concept for future preclinical and clinical work. Find Plasmids for Disease Research...research, researchers are eager to apply it in a clinical setting. CRISPR is often used for animal germline...exon skipping is a good option for DMD treatment. Clinical trials have used oligonucleotide exon skipping...rate of exon 51, which resulted in appropriately localized dystrophin. In a subsequent study, they used CRISPR...six months. Future directions and obstacles to clinical CRISPR editing Given the success of the CRISPR-mediated...be evaluated separately, increasing the time to clinical approval. Even with the challenges precision editing...become one of the first diseases to be treated clinically with CRISPR. Many obstacles remain for CRISPR...

...swimming where they periodically beat their tails left and right in a symmetrical motion to move forward...behavioral studies. In neuroscience, opsins are typically ion channels that allow charged particles to cross.... The non-specific cation channel ChR2 and the calcium channel LiGluR both cause ion movement that results...Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement unique to spontaneous slow forward... photostimulation in order to drive the full symmetrical response.  The function of Kolmer-Agduhr cells...Aristides B., Filippo Del Bene, and Herwig Baier. "Optical control of zebrafish behavior with halorhodopsin...

...to mind when we think “biomedical research". The family entered the biomedical field’s spotlight after...ways these animals have impacted humans through biomedical research. The Camelid family is composed of ...chromobodies,” which are single-domain antibodies genetically fused to fluorescent proteins and used to visualize...therapeutic was approved in 2018, with many others in clinical trials (Jin, et al. 2023). In addition to therapeutics...) (Xu, et al., 2021).      Figure 2: Genetically modified mice offer an alternative method to ...Arbabi-Ghahroudi M (2017) Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front Immunol...

...strengthened my critical thinking and problem solving abilities, increased my technical know-how, and helped...scientist.” For jobs away from the bench, look for “technical or product support specialist,” “field application...application scientist,” or “scientific or technical writer,” among others. Don’t discount job postings looking... 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...from the lab to a desk, although there’s often technical career tracks for PhDs who want to stay at the...

...Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate 14 of the 16 non-visual ...TRUPATH collection allows researchers to measure the physical association of combinations of different Gα, Gβ...acceptor. TRUPATH uses an updated version of BRET called BRET2. Figure 1: TRUPATH measures heterotrimeric...N-terminus of the protein. For the Gα constructs, localization of the Rluc8 was more challenging. The Roth ...experimental refinement to determine the optimal localization of the Rluc8 insertion within the Gα. Despite...transducers and that the potency and efficacy is typically non-uniform across the transducerome for a specific...well known and, as a result, a target of many pharmaceutical drugs, there is still only a partial understanding...

...generally linked to dopamine (DA), a well-known reward chemical and neurotransmitter. But aside from DA’s famous...specific dopaminergic neurons in a brain region called the substantia nigra, which is the main source ... it can be performed in a live awake animal by surgical implantation of the probe into a specific brain... animals (Patriarchi et al., 2018). Similar to calcium and glutamate sensors, the new dLight1 sensor is...in freely moving mice Record DA dynamics of optogenetically activated and inhibited neurons in live mice...neuronal imaging of dopamine dynamics with designed genetically encoded sensors." Science 360.6396 (2018): eaat4422... PMCID: PMC6287765. Sun, Fangmiao, et al. "A genetically encoded fluorescent sensor enables rapid and ...