...Verkhusha VV. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat Methods. 2011 Jul 31;...proteins Newly developed photoconvertible FPs like PsMOrange and mEos add another layer of complexity to imaging...GFP spreads through the cytosol via cytoplasmic strands. Image from Sattarzadeh et al., Sci Rep. 2015 CC...most useful for labs that already have created a transgenic GFP-containing organism,” says Hanson, “using...unexpectedly photoconvertible GFP proteins to make a new transgenic line could avoid problems in localization that...

...contains the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs...glycosylate proteins and may recapitulate the post-translational modifications added to proteins in other eukaryotes...includes a special feature for difficult-to-purify membrane proteins. pCri vector pCri-13a, designed for use...protein tag. MISTIC drags tagged proteins into the membrane and may improve expression of proteins that natively...

...proteins, or transcriptional regulators. In this system, Cas9 generates a double-stranded DNA break at...are complementary to either side of the double-stranded break in the genomic loci of interest. As the ... Find the CasX plasmids Drug inducible transcriptional activation and genome editing Yu Wang's lab...developed drug inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9...library contains ~93,000 unique gRNAs that target random positions along the E. coli MG1655 genome with ...

...visualize individual/non-overlapping cells. While transgenic animals are commonly used to specifically manipulate... to trigger recombination and expression of a transgene (Lin et al., 2018). However, this system requires...tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly...site to cleave and inactivate any read-through transcripts of the effector gene. Effector genes, any gene...across cell types and organisms by replacing the transcription factor binding site and effector in the construct... the SPARC system to use other experimental transcription activation systems, such as LexA/p65 or mCD8...

... gene, enhancer, and promoter – all rely on the random integration of a reporter construct in close proximity...integrate within an exon of a gene to generate a transcriptional fusion. This may disrupt the endogenous function...spliced into the gene product resulting in a transcriptional fusion, much like promoter traps. While this...likely disrupt the gene it monitors due to transcriptional fusion.   Examples of promoter, enhancer...monitored genes and mutants - viral integration, transposon, and many more! Check out Addgene’s catalog to...resources on the Addgene blog Plasmids 101: Using Transposons in the Lab Plasmids 101: Screens vs. Selections...

...). If you can get away with generating transiently transfected cells, you will not need a mammalian selection...mammalian selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent...create a new strain with a disrupted gene by transforming wild-type yeast with an integrating vector expressing...majority of fly vectors are based on p-element, random or targeted, integration into the fly genome and...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...

...positively transfected. Alternatively, you can use a selection marker to select transfected cells (for...nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does. There are ... The human U6 promoter prefers a 'G' at the transcription start site to have high expression, so adding...Before starting your experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive...resistance cassette, such as PX459). After you co-transfect the CRISPR/Cas system with your homologous recombination...

... charged ions to rapidly pass through the cell membrane and initiate an action potential - a very useful...and close when exposed to light and change the membrane potential. OptoXRs are modified GPCRs. When exposed...downstream signaling pathways that can change both the membrane potential and a host of other cellular functions...activity upon exposure to light within a specific range of wavelengths. Excitatory channels allow positively...Photoswitches are great for teasing out important branch points along a signaling cascade that may go awry...optimal wavelength to use but can be activated by a range of wavelengths. While this may give you a little...Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proceedings of the National Academy of...

...orthogonal attB-attP pairs per recombinase may be arranged on a register (Fig. 1C below). If attB-attP pairs...approach is to interrogate state with qPCR. The re-arrangement of DNA segments in each state of a register enables...sensors do not necessarily have to be based on transcriptional regulation, as long as they can control recombinase...recombinase activity. For example, post-transcriptional recombinase activation via RNA toehold riboregulation...expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to record all permuted... of a set of chemicals. Specific examples of arrangements that should be able to record all permuted substrings... this is to prevent interference between the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead...

... our values of transparency, reproducibility, and community-building. The transparency begins with sharing...Addgene’s antibody resource is funded by a generous grant from the NIH’s Brain Research Through Advancing ...share plasmids with each other. Together, we can transform the antibody sharing landscape as well.     References... Improving Reproducibility through Rigor and Transparency of Experimental Methods. Neuron 90(3):434-6.... project is supported by a NIH BRAIN Initiative grant (U24NS119916 ). The content of this article is solely...

...protein (LanYFP) from the marine invertebrate Branchiostoma lanceolatum in 2012. Using structural modeling...mNeonGreen can be used as a fusion tag for a wide range of applications from traditional imaging to FRET...Alberta and University of Oregon created mTFP1 by transforming E. coli with a gene library of approximately...mTFP1 can be used as a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters...particularly with fluorophores that excite in the violet range. For example, mWasabi can be used with Sapphire ...monomeric green fluorescent protein derived from Branchiostoma lanceolatum." Nature methods 10.5 (2013): 407...

... AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral vectors ...to be due to the viral particle’s affinity for heparan sulfate proteoglycans on the surface of the packaging...supernatant, the viral particles associate with the cell membrane and are taken into the cell via endocytosis. In...looking to improve their AAV yields. Given the wide range of downstream applications scientists rely on our...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show that there...test the endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe...

...postdoctoral fellows (PFs) face a number of challenges ranging from long periods of training to limited job opportunities...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...your own lab and get an independent investigator grant (e.g. an R01 from the NIH, in the United States)...postdoctoral mentor. A good mentor typically writes the grants that fund your research, guides the research directions... available for mentees due to travel, teaching, grant writing, etc. Also, it is unlikely that a mentor...reference, informal recommendations, more positive grant application and manuscript reviews, etc. You'll ... Non-Academic Career Decisions Check out our transferable skills guide Resources on Addgene.org Learn...

... method entails arranging all calculated p values from smallest to largest and ranking them (1 being the...dispersion – standard deviation/error, variance, data ranges. how closely data points fall to each other (see...significance by finding the largest p value where its rank divided by the total number of comparisons performed...– Tukey’s test, Newman-Keuls, Duncan’s multiple range test, and several others. Each test has different...negatives, moderate false positives. Duncan’s multiple range test – Most powerful. Low false negatives, high ...): 126-130 (2012). 10.1097/SMJ.0b013e3182498ad5 Curran-Everett, Douglas. Explorations in statistics: statistical...

...that green fluorescent protein through transcription and translation. Unfortunately, having live cells in... like gel electrophoresis to separate DNA or transforming cells with green fluorescent protein, these ... into a host cell (through a process called transformation), essentially giving the cell all the instructions...enzymes that can convert certain chemicals into fragrances that they can smell, or enzymes that can create...perhaps consider using some elements of BioBits to transition your undergraduate students from the classroom...

...-mAbs. It was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas...heavy (blue) chains as driven by two CMV promoters (orange). Figure from Andrews, et. al., Elife  A limitation...IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target antigen ...the restriction enzyme BciVI was present in the aberrant light chain sequence, but was predicted to occur...express multiple subclasses of IgG antibody. Broad range secondary antibodies are commonly used in research...

...Wen et al., 2018). SRIRACCHA uses the Piggybac transposon to stably integrate a reporter gene that contains..., you’re ready to edit. These cells are then transfected with several different constructs for the reporter... gRNAs by simply changing the gRNA used when transfecting your cells.  A plasmid that contains Cas9. The...plasmid.  With this set up, all successfully transfected cells will express RFP while cells containing... Lastly, once a mutant is identified, the PB transposase can easily and seamlessly remove the integrated...Knudsen C, Ásgrímsdóttir ES, Rahimi K, Gill KP, Frandsen S, Hvolbøl Buchholdt S, Chen M, Kjems J, Febbraro...

...let the team detect its co-localization with a membrane-localized mCherry or GFP protein. With this system...bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to...proliferation and differentiation.  Since Ras is a membrane localized GTPase, the team fused a mCherry OptoNB...OptoNB-SOS fusion protein in cells that expressed membrane-localized mCherry. With darkness, SOS binds Ras...using a fluorescent reporter for Erk kinase translocation. These results demonstrate that OptoNBs can ...nanobodies to control protein activity Read about RANbodies, reporter nanobody fusions Learn more about using...

...the delivered cargo (often a gene) is called the transfer plasmid while the other plasmids with packaging... or something similar, and is contained on the transfer plasmid. If expression is the goal, a promoter...Pol, the last component, contains the reverse transcriptase and integrase components to convert the cargo...other to allow DNA synthesis of the complementary strand to yield dsDNA. Unlike LTRs, ITRs are perfectly...delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry The viral packaging...the primary viral polymerase. E4/E1A activate transcription of other relevant viral genes (including the...

...times have you looked at a diagram depicting transcription, or DNA repair, or replication, or any number...researching enhancer elements that control the transcription of a gene a megabase away, or looking at the...the histone methylation status at a double-strand break that needs repairing. They may be looking at chromatin... protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous... lymphocyte receptor signaling. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. ...Mar 27;33(6):717-26. Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical...

...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher... of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for multiplexed...

...community. As a result, I have occasionally pondered translating my research into a startup company. However, ...school and turn it into a biotech company. Transitioning an idea from academia to a startup Mike Koeris... biotech startup, Mike explained, his career transitioned away from the bench and became all about finding...stages in the startup". To gain insight into "the random-walk" of a budding startup, I asked Mike to outline...company may begin to prove the technology. This transition takes around 1 – 2 years. The product is the...into it.” Have any other helpful tips to help transition a project from academia to a biotech startup ...

...interaction (PPI) studies in plants that rely on transient transfection systems using Agrobacterium (such as Nicotiana...indicator. Archon1 can embed itself into the cell membrane where cell voltage can then be measured. Archon1...also showed that Archon1 is functional in a wide range of animal models such as mice, C.elegans, and Zebrafish...al., 2016)). During Agrobacterium-mediated DNA transfer, each GOI is usually expressed from a different...plasmids to study PPI using Förster Resonance Energy Transfer (FRET, Heckler et al., 2015) or ratiometric Bimolecular...

...microenvironment. This results in increased expression of transforming growth factor-β signaling, possibly inducing...let’s take a look at exosomes. Proficient in transportation, exosomes are tools of communication and provide... (micro RNA) and other molecules that can be transferred to other cells, inducing epigenetic changes in...in targeted cells. In this case, exosomes may transport miRNA from drug-resistant cells to sensitive ones...enzyme that controls apoptosis and regulates transcription. Without TKA1, cells proliferate and cannot .... "Intercellular nanovesicle‐mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular...

...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, Ramanathan... include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA bait). ...changes in RC2H1 protein expression and post-transcriptional regulation of SM1v1-containing RNAs could play...often trick host proteins into binding their untranslated regions (UTRs) to aid in the viral life cycle...IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding proteins...

...help with transitioning out of academia (because so many scientists are making this transition). Another...have experience and knowledge to help scientists transition out of the academic sphere into a different meaningful...not always as effective in mentoring for this transition. Second, a coach will help you set goals and ...scientific backgrounds. For a stated purpose of transitioning a scientist from academia to industry, you might...in coaching for communication skills. Industry transition is also an area of specialty for Michelle Capes...

...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical... the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent change in...sensors available that will localize at the plasma membrane. GCaMP-X is a nucleus-targeted GCaMP6 with an ...an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing...

...chromoproteins are just now being developed for a broad range of biological inquiries.  What is a chromoprotein... a gene encoding a blue chromoprotein, after transforming them into your cells, you can simply look for... plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue...Image: Murakoshi et al., 2019. ShadowR is an orange light-absorbing chromoprotein that Hideji Murakoshi...imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) in living cells (Figure 4). They created.... K., Tietscher, S., Edlund, A. D., Jamal, S., Wistrand-Yuen, P., … Forster, A. C. (2018). Engineering...

...patients. And, to be honest, I’ve never enjoyed grant-writing. The more I talked to people with industry...only help. Like any major life decision, the transition felt scary. While I knew two people from graduate... to enter academia, it is easier to make that transition from an academic setting—especially if job applications...pages. In academia, however, writing papers and grants is an inescapable and essential part of being able...’re often required to at least attempt to write grants. If you don’t enjoy or excel at writing, an industry...postdoc will free you of the never-ending cycle of grant-writing that is academic life. Of course, because...

... non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20 nucleotide targeting...of wild-type Cas9 and a gRNA generates a double-strand break in the target DNA, which, when repaired through...scaled up for genome-wide screening due to the broad range of potential target sequences and ease of generating...heterogeneous population of gRNA-containing lentiviral transfer vectors, each targeting a specific gene within.... Pooled gRNAs are then cloned into lentiviral transfer vectors to create the CRISPR library. CRISPR libraries... you can skip the steps above! Cells are then transduced with lentivirus containing the CRISPR library...

...in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive... is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees... available, cost-effective, timesaving for transformations. Cons: Prone to satellite colony formation ..., interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or...selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes...confers mammalian resistance to G418. Con: Slow transformation   Spectinomycin/streptomycin Spectinomycin...

...2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was annotated in several genomes and is classified... Cas9’s shortcomings - namely its blunt double stranded cleavage and G-rich PAM requirement. Zetsche et...one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid...pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning... cloning. Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells...exciting both in terms of basic science and translational applications. This discovery of this type V ...

...aims do not need to be grant aims. Each aim could be the basis of a single grant. Either way, think realistically...talk could be your plans for your first NIH R01 grant proposal or a 10-year research plan. I suggest preparing...is a significant need for healthy livers for transplants. Growth of new livers in culture could resolve..., neither of these can substitute for a liver transplant. My lab will combine bioengineered scaffolds ... for presenting a science talk Check out our transferable skills guide Read all of our career blog posts...

...about? In March 2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA,...postdocs in the final ruling was, for a lot of us, frankly quite surprising. Consortia of higher education...issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions... of postdocs at different points on this salary range. We have been able to gather data on changes to ... raising salaries. In addition, both Brown and Brandeis universities have stated that they consider postdocs...

...project to the NAc, where enhanced glutamate transmission leads to locomotion sensitization. (for review... Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC projections to the NAc and decreased...injected Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression...the animal behavior. With the availability of transgenic animals and viral vectors, scientists are combining... they injected AAV2 particles into a Drd1-Cre transgenic mouse to deliver Cre-dependent expression of  ...Alpár, Alán, et al. "Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon...

...excellent choice for fusions with integral membrane proteins and membrane associated proteins (i.e. GPI-anchored...these compartments undergo significant post-translational modifications including glycosylation, disulfide... addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...including the secretory pathway (Figure 2), the inner membrane space of mitochondria and chloroplasts, the extracellular...oxidizing environments when targeted via the Sec translocon." Traffic 12.5 (2011): 543-548. Pubmed PMID: ...

...plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one... areas of microbial research like metabolism. Transport, biofilms, and much more. Because of it’s BSL-...have created molecular biology tools to generate transgenic axolotls for an array of different studies. Elly...popular tools for studying frogs include egg transplants and microinjections. Injections of mRNAs, for...Arabidopsis. There are also important tips for plant transformations and the use of Arabidopsis in the classroom...

...purification of tagged transcription factors in mammalian cells and transgenic mice.” PNAS 100.13 (2003...and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids to produce...lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the general process by which...is responsible for this highly specific post-translational protein modification. Most organisms have less...enzymatic biotinylation of proteins: A post-translational modification of exceptional specificity.” Trends...

...size, multimers may exhibit reduced transformation and transfection efficiency compared to the monomeric... Elfinger M, Hartl D, Rudolph C. Transgene expression of transfected supercoiled plasmid DNA concatemers...plasmid multimerization is to avoid growing the transformed bacterial culture into the late stationary phase...the linearized band, ligate the plasmid, and re-transform the ligated plasmid to recover the monomeric form...

...Impressions of NgAgo In particular, we need to thank Pooran Dewari who wrote this month’s most popular blog...NgAgo genome editing up and running in their labs. Pooran surveyed a CRISPR google forum to get a better ...CRISPR-based technique to generate C->T or G->A transitions in a target sequence in live cells. This technique...endonuclease (dCas9) fused to a deaminase to make the transitions at target sequences specified by gRNAs. Caveats...Epigenomics 5 Tips for Troubleshooting Viral Transductions Single Base Editing with CRISPR Cas9 Activators...