...tweet URLs with scientific domains <10% of the time. Figure 3: Top 20 Domains Tweeted by Scientists. 6....scientific domains more often than other scientists   Figure 5: Portion of Tweets with Scientific Domains, Categorized.... The #1 (re)tweeted domain by scientists was Instagram Social media and news sites were also popular....6. The top scientific domains tweeted by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific...Scientific American Figure 4: Top Scientific Domains Tweeted by Biologists. 7. Biologists, neuroscientists ...(orange box) post tweets containing scientific domains more often than other disciplines. The majority...tweets for most disciplines contain scientific domains between 0-10% of the time; however, ~20% of life...

...editor can install all possible point mutations, delete short stretches of DNA and even insert new DNA at...advancements and the noteworthy work of fiction that it inspired. CAUTION: Spoilers alert! Pre-DNA Era  H.G. ...enzymes, meganucleases, were discovered. These proteins are characterized by large DNA recognition sites... successful in the box office, indicating the insatiable appetite of the moviegoers for dinosaurs wreaking... In this movie Ethan Hawke’s character revolts against class divide that is created by genetics.    Zinc...DNA-cleaving strategy similar to the ZFNs, but instead of recognizing nucleotide triplets, the components...target new genomic sites. The discovery of the Cas proteins in 2005 was a major breakthrough in the world ...

...encoding the insert of interest as well as sequences necessary to encapsulate that insert into a viral... psi signal, the genes encoding the structural proteins could not be integrated into the new viral particles... one with the exception that the HIV accessory proteins that are not essential for the production of the...its activity. A CMV or an EF1α promoter has been inserted in this LTR so you don’t need Tat to transcribe...RCL) Efficient gene transfer Potential for insertional mutagenesis: Replication-incompetent lentiviruses...human tropism can still infect the researcher and insert into her/his genome - this is a biosafety risk ...dividing and non-dividing cells No immunogenic proteins generated Stable integration into host genome...

...Director of the Merkin Institute and Vice-Chair of the Faculty at the Broad Institute; Professor of Chemistry...Harvard University, and David R. Liu from the Broad Institute and Harvard University. Over the course of the...papers (data was provided by Addgene on 5/26/20 and contains requests from 5/22/14 to 5/19/20). Each paper ...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on... analytical expression for the data likelihood contains an intractable integral of the product of a Poisson... least impacted. These results highlight the instability and uncertainty in estimating parameters from...important additional caveat is that our dataset contains plasmid orders from just one lab, which limits...

... antibody against PARIS, a zinc finger protein involved in neurodegeneration in Parkinson’s disease. Knowing...rather than producing and purifying it in your lab. Instead of starting a new search or clicking through to...when we changed from searching for viral preps instead of plasmids? The available filtering options now...types of searches including depositing labs and institutions. For example, search for “Melina Fan” and you...Flame is more than an award for depositors and inspiration for our updated mascot, it’s also a convenient...Species: Specify the source organism for a gene insert. (e.g. S. cerevisiae) Plasmid Type: Choose an empty... empty backbone if you intend to clone your own insert, or use the other options to find plasmids matching...

... is present even in common lab cloning strains. This intrinsic bacterial recombination pathway, referred...region (calculated using Oligo Calculator). Insertions - To insert short sequences of up to 100 bp, such as...referred to as recA-independent recombination, joins together pieces of linear DNA through short homologous...recombination reported in all laboratory E. coli strains tested so far. The use of this pathway as a cloning...the PCR mix to remove parental DNA (37 °C for 15 mins). This enzyme specifically cuts methylated DNA, ...used to perform any plasmid modification, from insertions, deletions, and point mutagenesis to complex ...template binding regions to amplify away from the insertion site. Encode half the new sequence at the 5’ end...

...to modified proteins called histones. We can detect modified histones with “GPS for proteins,” ChIP-seq... coordinates: chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the...techniques Technique What It Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq ...For details on DNA compaction and the structural proteins that make it possible, see the appendix DNA is... you run a ChIP-seq experiment (think “GPS for proteins”) for NANOG, an essential transcription factor...epigenomics: H3 – one of NANOG’s associated histone proteins K4 and K27 – locations of the amino acid lysine...enhancers, lncRNAs, repetitive elements, repressors, insulators, and more. We know that this matter exists, but...

...locus-specific ChIP). These methods consist of insertional chromatin immunoprecipitation (iChIP) and engineered...interactions. For this purpose, we developed the insertional chromatin immunoprecipitation (ChIP) (iChIP) ...purification, in 2009. However, iChIP requires insertion of recognition sequences of an exogenous DNA-binding... DNA-binding molecule technologies such as TAL proteins and CRISPR became available, I was very excited...easily isolated for identification of associated proteins, RNAs, and other genomic regions. I believe that...genome functions in the hope of developing drugs against intractable diseases. Currently, a number of labs...analyze signal-induced nuclear translocation of proteins in 2004. In addition, we have recently developed...

...lines. Identifying less toxic envelope proteins or envelope proteins that target specific tissues are areas... lentiviral expert) Didier Trono's lab webpage explains how lentiviral particles are produced by co-transfecting...that's gag, pol, and env), the isoforms of these proteins, as well as the viral long terminal repeats (LTRs...envelope plasmid and a packaging plasmid encoding HIV proteins gag, pol, rev, and tat. 3rd generation systems...In addition the deletion reduces the risk of insertional mutagenesis by limiting the interaction between...components are HIV-based? A7: No single plasmid contains all the components necessary to produce viral ...vectors contain gag, pol, and rev. Second-generation contains those three genes plus tat. The envelope plasmid...

...segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression... Produce proteins that glow so that scientists can track their location or quantity inside a cell ...2014 and updated Apr. 14, 2020. Any newcomer who joins a molecular biology lab will undoubtedly be asked... a plasmid by recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection... the MCS is often downstream from a promoter. Insert Gene, promoter or other DNA fragment cloned into...commonly referred to as “vectors” or “constructs.” To insert a gene of interest into a vector, scientists may... are complete, the vector containing the newly inserted gene is transformed into bacterial cells and selectively...

...Place intended insert in between nick sites Use 40 nt homology arms for small insertions or tags (ssODN...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create... creates a non-target strand-cleaving nickase. Instead of cutting both strands bluntly with WT Cas9 and...deletions, while H840A is biased towards large insertions. These profiles are likely due to the distinct...can’t find a good gRNA that cuts close to your insertion site, you can’t obtain high HDR efficiency. Nickases...and Yan designed a PAM-out nickase experiment to insert an EcoRI site using a single-stranded oligonucleotide...oligonucleotide (ssODN) donor. Using a D10A nickase to insert an EcoRI site in various spots between the nick...

...proper folding of the tag, which prevents insoluble fusion proteins from being purified by GST (maybe try ...between – now all you need are some of your favorite proteins purified to carry out your plan. With a little...purification a cinch. Types of affinity tags, the proteins they work best attached to, and the species they...will be expressed along with all other cellular proteins within the expression system (bacteria, yeast,...tags also have commercially available antibodies, resins, beads, and other purification aids readily available...for detection and/or purification of MBP-tagged proteins. The antibody is an alternative to amylose resin...Nickel, Cobalt, Copper, etc.), allowing His fusion proteins to be purified by single step metal affinity chromatography...

...used for an array of detectable proteins including fluorescent proteins, antibiotic resistance, and luciferase...monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin...inactive mCherry, the lab introduced a 43 base-pair insertion that disrupts fluorescence due to a frameshift...TCA-to-TUA) found within the 43 base-pair mCherry insertion. Editing of these motifs generates lesions that... editing capability of the seven human APOBEC3 proteins, finding that APOBEC3A and APOBEC3bctd are the...populations. To develop this method the lab was inspired by work that used an integrated GFP reporter that...DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine at amino acid 66. This histidine is...

...vague and "what proteins regulate the Z pathway?" is an open-ended list of proteins. Instead, consider "how... at the National Institutes of Health, was an Associate Professor at Albert Einstein College of Medicine...Hughes Medical Institute. His interests include the quality control of secretory proteins in the Endoplasmic... Research Campus of the Howard Hughes Medical Institute. Are you currently on or planning to go on the..."how do proteins X and Y regulate the Z pathway?" A well-defined outcome makes it easier for the search...favor a different model. Finding evidence for or against a model is a standard scientific strategy. However...Endoplasmic Reticulum, optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also...

...making copies.     Types of origins of replication There are lots of origins of replication out there so...: The insert Bacteria tend to maintain fewer copies of plasmids if they contain large inserts or genes...elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must...known derivatives), pSC101, R6K, and 15A. Not all origins of replication are created equal. Some will produce...whether the ori is positively regulated by RNA or proteins, respectively. A plasmid's copy number has to ...mutations in the replicon. For example, the pMB1 ori maintains about 20 copies per cell, while pUC – which differs...toxic product. The E. coli strain Most E. coli strains can be used to propagate plasmids, but endA- E....

... reporter mRNA, which contains the tethering sites for the fluorescent proteins and is driven by an inducible... to monitor local translation and determine if proteins are targeted to their destination co- or post-...Translating RNA Imaging by Coat protein Knock-off) instead takes advantage of the ribosome’s movement. When... along an mRNA, it displaces other RNA-binding proteins to allow translation to occur. For a TRICK reporter...only a red FP bound to the reporter. Because it contains a nuclear localization signal, NLS-PCP-GFP then...mRNAs and their corresponding newly synthesized proteins. As in the TRICK system, the 3’ UTR of the reporter...labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind the mRNA to the plasma...

...majority of research institutes.  Pro tip! In most academic and nonprofit institutes, the Tech Transfer ...materials are requested, instead of coordinating signatures from two different institutes. Our online portal...commonly used ones for transfers between academic institutions.  What is the UBMTA, you ask? Well, organizations...signatory) to the MTAs for plasmids in our repository. Instead, we facilitate the signing of MTAs by the Provider...people think our MTAs are different than other institutes. But they’re not! The vast majority of our plasmids...that plasmid facilitated by Addgene — basically, instead of having to sign individual MTAs for each request...Recipient Scientist Acknowledgment (RSA), which contains the terms of use for the requested material. Once...

...product to insert, and tools for primer design, so it’s always best to check in with the instructions from ... Gibson and colleagues at the J. Craig Venter Institute in 2009. Here at Addgene we added this option ...fragments, but can also be used in basic cloning of an insert into your vector of choice as shown in the figure...incubated together with an enzyme master mix, which contains three different enzymes: an exonuclease, which...using Gibson assembly, imagine that you want to insert your gene of interest into a vector with a large...included in the vector you want to use. If you inserted these two pieces of DNA by restriction enzyme ...fragments. However, by using Gibson assembly, you can insert both the gene of interest and the tag sequences...

...allowing a spacer-repeat “brick” to be inserted. The BsaI site remains intact, allowing subsequent “bricks...make sure that each cell that gets the plasmid contains all of the desired gRNAs. This increases the chance...the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two U6 promoters. To use ...available from the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods....)-containing plasmids. Each destination vector contains GFP, enabling you to select cells with high GFP...enables users to express up to 7 gRNAs. The kit contains different destination vectors depending on the...through Golden Gate or Gibson Assembly. gRNAs are inserted into the pCBC vectors using BsaI, and promoter-gRNA...

...include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane...membrane proteins. Although all viral components are available in bacterial expression plasmids, glycoproteins...glycoproteins and other proteins that require post-translational modification may not be easily expressed ...level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility... doing things. These new processes have been instrumental in achieving our mission to help science move...Virus Entry Mediated by SARS Coronavirus Spike Proteins. In: Methods in Molecular Biology. Humana Press...

... 100 kD proteins or 20, 40, 60, and 80 kD proteins. Plasmids containing 150 or 250 kD proteins are also...expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins run as ladders...the Genetic Pertubation Platform at the Broad Institute have expanded the genetic screening toolkit by...express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli, either individually (~20,000 lanes...also available separately. The proteins, which are purified via histidine tags, are optimized for SDS-PAGE...Western blots via engineered immunoglobulin binding domains.  Santilli, R.T., et al. Sci Rep (2021). https... large size of CRISPR-Cas effectors and fusion proteins, such as Cas9 and Cas12a, creates challenges for...

...mice, exosomes expressed specific integrins (cell-adhesion proteins) targeting different organs. This ...action outside of tumor cells, and recent studies (Martins et al., 2018 and Xing et al., 2010) revealed precisely...physiology is deeply connected to these 3 stages and contains multiple cell types: fibroblasts, myofibroblasts...whole, microenvironments promote tumor durability instead of repressing them, making it even harder to cure...possibly inducing the secretion of growth promoting proteins, such as human growth factor, vascular endothelial...microenvironment itself. They deliver compounds like proteins and nucleic acids from cancer cells to the microenvironment...microenvironments! Take myoepithelial cells for instance. They retard tumor formation by inhibiting cell...

...recognizing the target protein remains bound to the column while the rest of the proteins are eluted. Then, the...full-length protein that you want to generate antibodies against, or a unique peptide sequence derived from this...the serum are separated, and the serum - which contains the antibodies - is further processed to isolate...Protein G purification is one common method. These proteins bind the IgG heavy chain. Running the serum over...specific to the immunogen or not, while any other proteins pass through. However, protein A and protein G...Antibodies that don’t cross-react with the serum proteins pass through the column, resulting in a polyclonal...or select a monoclonal or recombinant antibody instead. Otherwise, you might experience variability in...

...amount of information regarding which regulatory proteins, signaling molecules, mutations, and environmental...represented in a very small number of registers. Instead, one may use qPCR to determine the fraction of ...combination of red, blue, and green fluorescent proteins in each state. The three methods of state interrogation...tell you the distribution of states in a sample; instead, it will suggest the predominant state of registers...plasmid (high copy, ColE1 origin of replication), contains large serine recombinase genes downstream of inducible...copy, bacterial artificial chromosome backbone) contains the register. Depending on the desired application...endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to...

...Massachusetts Institute of Technology, Boston University, Johns Hopkins University, the Salk Institute, and the... the Karolinska Institutet, among others). Mentors and mentees held scheduled monthly progress video conferences... confined by birth country or financial means. Instead, talent should be realized through equal opportunities...internationalization faces several obstacles; for instance there are few resources to help get individuals...supported, and can more fully identify with and be inspired by the mentor’s successful example. For evidence...2014-15, University of Valencia): “It is very inspiring to be in contact with mentors who are at a professional...universities and high level public and private institutions also showed their support and hope for the IMP...

...sharing their worm strains through the Caenorhabditis Genetics Center (CGC) which maintains and distributes...each half to a gp41-1-N-intein. Split inteins are protein domains that naturally “associate, self-excise...distributes the strains all over the world. When I was at the 22nd International C. elegans meeting, I was again... the Arizona State University (ASU) Biodesign institute developed a 3’UTR database specifically for C.... discovered in plants to conditionally deplete proteins of interest in C. elegans (Zhang et al., 2015)...modified TIR-1 protein can degrade degron-tagged proteins in an auxin dependent manner. This system allows...scientists to both spatially and temporally knock-down proteins of interest.  Plasmids for cGAL and split cGAL...

...long ncRNAs is impractical. Instead, tags or stop elements can be inserted to disrupt the transcription...RNAs (ncRNAs) are transcribed but do not encode proteins. They play important roles in many cellular and...permanently disable, protein-coding genes by generating insertions or deletions in the DNA sequence. This same approach...break works well because very small deletions and insertions in the DNA sequence can completely disrupt expression... an open reading frame, so small deletions or insertions may not result in a functional loss (Ho et al...the ncRNA (Hazan and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription...exon or a large genomic fragment. (D) Knock-in: insertion of a synthetic polyadenylation (spA) signal. Figure...

...AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific AAV delivery Recombinant anti-FLAG...COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under CC-BY...for your next experiments, they will also likely inspire further-improved versions. Thanks to Christophe...Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this section.  Find mStayGold plasmids...2023). Bright and stable monomeric fluorescent proteins derived from StayGold. ResearchSquare (Preprint...alchemy: CAP-Mac unleashes the secrets of primate brains with brilliance By Hridaya Patel (Addgene co-op...systemic gene delivery in non-human primate (NHP) brains.  Systemic gene delivery capsids previously identified...

...PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection by Joachim...optogenetics, chemogenetics, plasmids, fluorescent proteins, careers, and more. These resources can now be...Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley, Shreya Vedantam, Michelle Cronin...used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi by Jennifer Tsang...and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for ...Fluorescent Vegetables in Aptamer Soup by Aliyah Weinstein RNA aptamers, which are nucleic acid sequences...shares some strategies for generating GFP fusion proteins. From considering protein size and shape, linker...

...plasmid's journey begins First, each package is opened and the contents are carefully inspected to verify that...different strains of E. coli that can be used for transformation. The preferred E. coli strains for storage... then the journey of transformation and storage begins. Some samples are submitted as bacterial colonies...the year (see graph below). Addgene scientists inspect each sample and make sure they have been barcoded...assigned to a location in a metal freezer rack that contains five levels that can hold five plates on each ... back into because every barcode on every tube contains information about that tube’s appropriate location...been received and stored, it is put into E. coli strains that have been treated to easily take in plasmids...

...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...amino acids found in all proteins, so you can't distinguish the different proteins from each other. To distinguish...Lastly, there are fixable dye stains that react with free amines on and inside the cells and are thus typically...distinguish different target proteins in your flow analysis, you'll need to use fluorophores that attach...appropriate readout. A common method of labeling proteins is to fuse them with a fluorescent reporter, such... reporter's fluorescence. Another way to label proteins is through treating cells with fluorophore-conjugated...antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow cytometry experiment are known...

... of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural Research Program (NIH-NIDA...are numerous variants for the different types of opsins available and their differences and uses have been...discussed thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University...details about types of constructs available, instructions for obtaining a material transfer agreement,...published at this point.  In brief, some standard opsins (see table above) include Channelrhodopsin2 (Chr2...and subsequent firing of an action potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride...specific opsin expression. However, the types of opsins you are able to use are obviously limited by the...

... Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 is an enormously...and former researcher at the Wyss Institute for Biologically Inspired Engineering.     Alejandro (Alex)...prevent your gene from being up-regulated. For instance, generally, the more highly expressed a gene is...similar tools were made using other DNA binding proteins such as zinc fingers (ZF) and TAL effectors (TALE...scale CRISPR-based screens can be used to gain insight into protein structure and function (Pan et al....Ansari R, Harper S, Jackson DA, McRae R, Pooley R, Wilkinson P, van der Meer D, Dow D, Buser-Doepner C, Bertotti...Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated...

...microbial strains. It proved to be impractical to catalog every variant of a feature. Instead, we identified... days or even weeks. Primer design was done painstakingly by hand. Records of plasmid construction were...their goals as intuitive and painless as possible. Instead of crowding the interface with every possible option... antibiotic resistance markers and replication origins, but there is extensive heterogeneity in the feature...efforts, such an algorithm has limitations. For instance, it could miss a common feature if the sequence... inaccurately. The best example is fluorescent proteins, which often come in closely related versions ... genomes of newly sequenced bacterial or viral strains. We are working on a way for SnapGene users to ...

...and find an ideal antibody against Protein A and an ideal antibody against Protein B. The antibodies are...isotype? Antibodies are composed of light chains and heavy chains made up of variable regions and constant...interact. You read extensive research on the two proteins and come up with a great experimental plan that...detect Protein B and successfully visualize both proteins.     Figure 2: Two primary antibodies ...In most cases, the resulting chimeric antibody retains the binding specificity to the original target ...target and can be used with secondary antibodies against the new Fc region. In addition to increasing multiplexing...     Morelock, M. M., Rothlein, R., Bright, S. M., Robinson, M. K., Graham, E. T., Sabo, J. P., Owens, R.,...

...transgene.The AAV encoding part A of the transgene contains a promoter and the 5’-fragment of the transgene...donor. The AAV encoding part B of the transgene contains a splice site acceptor followed by the 3’-end ...methods. The AAV encoding part A of the transgene contains the promoter, the 5’ half of the transgene, a ...homology. The AAV encoding part B of the transgene contains the region of homology, a splice acceptor sequence...help prevent the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008 used a...directed repairs (HDR) with CRISPR/Cas9. Donor A contains: 400bp homology arms to the site in the genome...stuffer DNA after the gRNA target site. Donor B contains: 400 bp homology arms to donor A,and “part B” ...

...apolitical process for seeking knowledge. The process begins with a testable hypothesis - an educated guess ...scientists then revise their hypotheses and the cycle begins again. No part of this process is inherently political...States government? Politics and science are intrinsically linked: governments exist in reality and science...Environmental Protection Agency, and the National Institutes of Health endanger scientists’ ability to uphold...consequences While frustration and anxiety have inspired people to rally for science, others are concerned... themselves can come to conclusions themselves instead of depending on scientists or politicians to interpret...lines (18). Feeling like an apple will fall up instead of down when dropped will not make it happen. In...

... generally preferred for large insertions such as fluorescent proteins or selection cassettes. This class...conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Quadros RM, et...Stroik January 24, 2023. DNA damage drives genome instability and contributes to cancer, premature aging, and...sticky or blunt ends and often introduces small insertions and deletions along the way. HDR uses homologous...overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for ...template to create mutations HDR donors can contain insertable sequences or specific mutations, along with homologous...target sequence to be modified. In general, the insertion sites of the modification should be very close...

...Plasmids expressing fluorescent proteins will be used to tag individual strains of bacteria. Their interactions...diabetes mellitus. Recombinant human proteins that play a role in insulin regulation and diabetes will be ...and the activity of the plant compounds on these proteins will be tested. Investigating changes in neuronal...after ischemic brain injury Dr. Gohar Tsakanova, Institute of Molecular Biology of the NationalAcademy of...condition is crucial. Plasmids expressing fluorescent proteins and a new two-photon laser scanning microscope...manipulate neural populations in order to get new insights into functional network interactions. Combining...bacterial strain. The sensor will be transported inside a small 3D printed robot that will “swim” in the...

...binding with Rep proteins and mutations in the trs site reduce Rep nicking. Since Rep proteins are important...with the viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication... with ITRs that flank the gene cassette, which contains a promoter, the gene of interest, and a polyA ...flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide Rep binding element (RBE) that ...resolution site that serves as the target site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type...type ITR in flip and flop orientation. The ITR contains three palindromic sequences (A-A’, B-B’, and C-C...sequences. Using recombinant-deficient bacterial strains such as NEB Stable, Stbl3, JC8111, SURE2, or XL10...

...Alex Chavez, researcher at the Wyss Institute for Biologically Inspired Engineering and member of the Addgene... vectors (~8.5 kb of insert) and the reduced titer associated with large inserts can make lentivirus production...particular, the Church lab at the Wyss Institute for Biologically Inspired Engineering and the Zhang and Doench.... For example, we’ve teamed up with the Broad Institute to provide lentiviruses for delivery of SpCas9...already expressing Cas9. As John Doench (Broad Institute and member of the Addgene Advisory Board) says...Doench/Root labs from the Broad Institute for providing us with advice and expertise as we’ve gotten these...

...exists to prevent substances in the blood, like proteins and viruses, from crossing into the brain. While...increase the chances of inducing an immune response against the capsids or transgenes. Sensory neurons and ...as the type of transgene delivered: fluorescent proteins, optogenetic sensors, CRISPR elements, etc... ...capsids express differently in different mouse strains, a phenomenon that we are calling “strain-specific... but is more effective than AAV9 in many other strains, including FVB/NCrl, 129S1/SvImJ, DBA/2J, and C57BL...table from Huang et al, 2019 for affected mouse strains). However, other capsids cross the BBB via interactions...interactions with LY6C1 are effective in both mouse strains. It is therefore important to review the literature...

... a VSV-G envelope expressing plasmid. Envelope proteins determine which types of cells a virus can infect...invariant sgRNA scaffold and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows...pCMV-VSV-G was deposited by the Bob Weinberg lab and contains an envelope protein for producing lentiviral and...lentiviral packaging plasmid from the Trono lab contains the rev gene. It’s meant to be used with pMDLg...lentiviral packaging plasmid from the Trono lab that contains the gag and pol genes. It also requires pRSV-Rev...lab is a 3rd generation lentiviral vector that contains a scrambled shRNA. This scrambled shRNA is used... cloning vector. Tet-pLKO-puro - Tet-pLKO-puro contains all the necessary elements for packaging, reverse...

.... Antibody heavy chain proteins as well as the genes that encode those proteins are designated by the ...antibody isotypes. Binding to complement system proteins, which can initiate a cascade that helps attack...their own. The antibody’s heavy-chain constant domains, or Fc region, can be bound by special receptors...location of disulfide bonds, the number of constant domains, and the hinge region length. Now for some essential...minor differences in their heavy chain constant domains. These are typically numbered, i.e. IgG1, IgG2,...IgG3, and IgG4. IgE IgE antibodies help protect against parasites but also play a role in allergies. They... any of the other isotype heavy chain constant domains. This switching helps B cells adapt during the ...

...If you study proteins, you’re probably quite interested in the canonical 20 amino acids. But in your ... add in fluorescent or chemically reactive side chains, all of which can help determine structure and ...for the ncAA integrated into the cell’s genome, instead of incorporation via plasmid or media. Theoretically... systems – the cells growing and producing the proteins – are not, and many of the existing GCE tools ...recombinant expression of phosphoserine-including proteins in E. coli (Zhu et al., 2022). It uses the TAG...for the phosphoserine and, because it uses ∆serB strains, does not require exogenously added phosphoserine...checking out all of the plasmids and bacterial strains (available at Addgene for GCE work. Plus, keep ...

...However, there are instances where traditional base editors are preferred. For instance, if target nucleotides...genome editing technique that mediates targeted insertions, deletions, and all possible base-to-base conversions...pegRNA) that both specifies the target site and contains the desired edit (or edits) engages the prime ...combined with any of the PE2-PE5 strategies. PEmax contains a reverse transcriptase with a sequence optimized...strategies to evolve more effective prime editor proteins, but found that the mutations that emerged depended... PE6a and PE6b are small prime editors with RT domains derived from the E. coli Ec48 retron RT and the... ✓ ✓ ✓ Evolved reverse transcriptase domains Smaller size and/or improved efficiency   ...

...each mutated allele.  Cre-lox expressing strains Cre-lox strains are a common example of mice that are derived... Crossing mice to generate a desired phenotype Instead, genetic engineering should be used to separately...independently into different mice, and then these strains should be crossed to generate double-mutant offspring...experiments. You should also maintain your parental strains throughout this process. In the F1 and later generations...genetically engineered mice with a single mutation or strains that have been crossed, there are many considerations...gene randomly into their genome, some transgenic strains are difficult to breed. Some problems with transgenic...expression level of the transgene. In existing strains of mice, the transgene can be maintained on a hemizygous...

...and identified interactions of these proteins with human proteins. Their deposited plasmids expressed ...over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman ...infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published a protocol...receptor for SARS-CoV. The lab also found that cathepsins play a role in SARS-CoV infection and deposited...Biering et al., 2021). In their search, they found mucins have an important role in viral entry, which are...viral infection.  Expression of SARS-CoV-2 viral proteins In the early weeks of the COVID-19 pandemic, the...expressed 26 of the 29 SARS-CoV-2 proteins which all but one are cloned into pLVX vectors (for lentiviral packaging...

...Some bacteria possess plasmid-borne toxins like colicins and subtilisins to kill other bacteria in the area...microbial defense Genes allowing bacteria to survive toxins are the clearest example of a plasmid benefiting...Conveniently, plasmids carrying genes for bacterial toxins like colicin typically also carry the necessary... the host immune system, and those that produce toxins that cause disease. While these plasmids can be...DNA and other molecules to host organisms. For instance, vectors derived from the Agrobacterium Ti virulence... resistance to that toxin, CcdA. Knowing which strains of E. coli contain this F plasmid is critical for...about some other common plasmid features, such as origins of replication and promoters, check out our other...

...markers. Need fewer components? A spacer can be inserted instead, allowing everything to stay in the correct...spontaneous insertion)   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy insulator sequences... the right CRISPR vectors on Addgene and found instead ones that were… pretty close, or at least close...approach, it’s easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus...various delivery methods, along with compatible inserts. All destination vectors contain an ampicillin ...also why you can’t skip a module entirely, but instead will have to replace it with a spacer.) Each component...expression) Mammalian Plasmid pRDA_791 (contains only orio and ampR) Drosophilia Plasmid...