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...receiving the translation, Addgene contracted with a professional scientific translator, L. Denise Roche...101: Una herramienta de edición Addgene tiene una gran audiencia internacional, incluido trabajando con...101 eBook is now available in Spanish!  This translation was gifted to Addgene by Daniel Mejías Sánchez...Roche, to edit the translation. We are pleased to announce that CRISPR 101: Una herramienta de edición ...  Many thanks to:  Daniel Mejías Sánchez, PhD, translator  Denise Roche, PhD, MD, editor, ldeniseroche@...

... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...negative selection marker selects against improper, random recombination into a different locus. Correctly...contain the negative selection marker, but cells with random recombination may incorporate the negative selection...and animal models, their efficiency is very low, ranging from undetectable to 0.1%. In contrast, new genome...

...this surface”….where the complex is docked on the membrane of the endoplasmic reticulum. And graduate students...reported cleavage site on the complementary DNA strand. And this is why we love working with researchers...”. Jacob computationally added the non-template strand to the Jinek structure so that the resulting physical...protein must have in order to scan along the double-stranded DNA, recognize a PAM sequence, allow the complementary...pairing between the guide RNA and the target DNA strand, and then activate two different endonuclease active...structure captures the displaced non-target DNA strand and shows the HNH nuclease domain swung around ...juxtaposition to its cleavage site on the target DNA strand. In addition, this model also displays the several...

...over for other applications, like PCR, cloning, transfection, or long-term storage. But what about those ...inverted terminal repeats. We use NEB Stable to transform and prep high quality DNA that contains these ... in a protein expression strain, we suggest retransforming the plasmid into a strain meant for DNA purification...prepping. Be sure to stay within the recommended range of the filter or you might inadvertently lower the...concentration Low copy plasmids produce fewer transcripts of their antibiotic resistance genes and can ...for high copy plasmids in standard growth strains range from 12-16 hours, but cultures with lower copy plasmids...to Quantify Your DNA Learn to Do a Bacterial Transformation Learn How to Perform a Diagnostic Restriction...

...mammalian cells can perform human-like post-translational modifications that bacterial and most yeast ...be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain...they’ve been separated onto two. You’ll first transfect your cells with your plasmid(s), and then wait... from dropping the tube they are contained in (Randolph et. al., 2018). At Addgene, we ensure the efficacy... Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells...A. Pathak, Christopher J. Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization..., https://doi.org/10.1016/j.cocis.2014.10.002. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci...

...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target...deaminase ADAR2DD to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the ...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note...therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging the construct...

... that may have warranted their own chapters, and certainly chapters that could warrant their own books..., and he wisely makes no such attempt. He is transparent about his own biases--many of which will be shared...non-scientists, Zimmer’s observations will likely range from mildly surprising to genuinely shocking. Mainstream...he describes the now infamous tale of Rosalind Franklin and her essential-but-unlauded role in the identification...within this section. The GFP story has its own Franklin in Doug Prasher, who originally isolated the GFP...science produced by the quieter, more reserved Franklins and Prashers will find it increasingly difficult...

...expression by interacting with proteins such as transcription factors and chromatin modifiers. These proteins...at the promoter region of a gene to initiate transcription (Figure 1). Although all cell types share the...functional in every cell. Differential expression of transcription factors, which recognize and bind to different...capitalized on. Figure 1: Schematic of transcriptional regulation by an enhancer. Created with BioRender.com...Tasic, B. (2025). A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell... A., Bickmore, W., Dean, A., Nobrega, M. A., & Bejerano, G. (2013). Enhancers: five essential questions...nrg3458 Ray-Jones, H., & Spivakov, M. (2021). Transcriptional enhancers and their communication with gene...

...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...One can inject plasmid DNA in the eggs to get transgenic animals. They might be immortal. Manihot esculenta...ReMOT control system (Receptor-Mediated Ovary Transduction of Cargo)  delivers Cas9 RNP to the arthropod...

...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...addition of IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the... the expression plasmid in order to repress transcription from a hybrid promoter. Although the DE3/T7 ...

...the non-profit sector are transferrable to industry such as technology transfer, communications, medical... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an... facility management, collaboration management, grant administration, clinical operations, and so on. ...include evidence such as proof of US government grants that the researcher is working under in the lab...new accomplishments to add. Obtain copies and translations of documents that are not in English. Create...    Additional Resources on the Addgene Blog Transferable Skills Guide 6 Steps to Submitting a Resume ...

...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...Post: Trump administration tells EPA to freeze all grants, contracts The Guardian: Trump to scrap Nasa climate... Scientists Find a Voice at Massive Rally for Immigrants National Science Foundation: Universities Report...

...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants... organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is big GFP is a ...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells." Science 296.5569 ...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...

...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...required since the E. coli you transform your PCR products into will efficiently patch up the DNA. The ...parental plasmid provides a mean for selecting for transformants which have taken up the mutagenized plasmid....

...therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using ...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-cells...rates of off-target cleavage. In both cases, translating the in vitro work into an animal model comes ...cleavage. Despite the potential difficulties in translating these findings into a therapy, these papers present...

...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 ...

... 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...

... 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...

...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...

...). 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...

... 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...

... 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...

...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...

...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...

...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...

...-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...

...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...

...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...

...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...