...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...

... There are two methods of neuronal tracing (Fig. 1): one to visualize neurons that send information to...significantly updated in 2023 by Jason Nasse.  References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some viruses (e....e.g., rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but...efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality to...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate an...

...sticks out and can be considered a linker (figure 1). In fact, for several FRET biosensors (yellow cameleon...fluorescent protein are relatively close together (figure 1). Where should the fluorescent protein be inserted...fluorescent protein database." Nature methods (2019): 1. PubMed PMID: 30886412. Mastop, Marieke, et al. "A...et al., 2018). Unbiased, random insertions can be generated with a transposon-based strategy (Sheridan...fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience... This type of interference is known as steric hindrance. For instance, GFP may occlude catalytic sites...sites or motifs that are necessary for post-translational modifications. Despite these potential issues...

...blotting, named for its inventor Edward M. Southern (1), can be used to detect DNA identity, size, and abundance...hours at low cost with restriction mapping (Figure 1). Though some labs may prefer to send DNA directly... serotonergic system and behavior.     References 1. Southern, Edwin Mellor. "Detection of specific sequences...into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization of ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts... control sample. Southern blotting applications range from confirming the results of cloning or knockout...

...refreshed figures made using BioRender.com. Figure 1: PRIME editing Please check out the updated guide... to use CRISPR plasmids in the lab and the vast range of possibilities at their disposal. With this update...CRISPR’s ability to make large genome edits using transposases or the Cascade-Cas3 system. We also describe...

...restriction digest and colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation... doi: 10.1371/journal.pone.0038313. Epub 2012 Jun 1. PMID: 22675541; PMCID: PMC3365890....-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...

...research like metabolism. Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one...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...

...prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who believe...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the...with viruses or transmissible diseases, so we were left with no choice but to quarantine in our houses,...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...we call “asymmetric collaborations.” These are transient partnerships where a researcher makes a small ...blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...

...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ...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...

...gel image includes a 1kb ladder (lane 1) that has bands ranging from about 500bp to 10kb, with the 3.0kb...restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction enzymes which...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...

.... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such ... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...in our cells in the single strand form, but double stranded RNA and RNA:DNA hybrid molecules both exist...arise from a ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly occurring...

...endonuclease activity degrades the displaced strand.   Figure 1: EvolvR consists of an error-prone polymerase...mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M...lineage tracing cells that do not tolerate double-strand DNA breaks by introducing a unique tag into a genomic...

...always guaranteed. To learn more about this process check out our Mouse Modeling blog posts: Part 1: Genetically...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

...blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently induce expression..., they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...small, double stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression (more...LoxP vector.   Turn it off for a little while Transient suppression of expression is faster and easier...expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on forever Permanently...

...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process...Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete ...step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is...

...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and...and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components and...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...need a protease reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed...However, the lab wanted to increase the fluorescence range to better study the spatio-temporal dynamics of ...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...

...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...

... et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system. Image from Xiong...Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing...use of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et...“single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that can...protein by a specific transcription response element (TRE) or kinase translocation reporters (KTRs). Find...interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism, the...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova... engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...