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  1. Treating Muscular Dystrophy with CRISPR Gene Editing

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    Blog Post
    ...point mutations. In addition to upregulating HDR, NHEJ must be downregulated to prevent the introduction ...short period of time, which would prevent the accumulation of unwanted mutations over time. DMD is a good...The homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping... when the genome editing frequency is low, the relative abundance of exon-skipped mRNA is high, likely...ataxia telangiectasia, congenital disorder of glycosylation, and Niemann-Pick disease type C. Although the...precision editing tailored to smaller patient populations. To bring DMD CRISPR therapy closer to the clinic...challenges above and more. One chief challenge is upregulating HDR in mature tissues, as this process is necessary...

  2. Fluorescent Proteins 101: Fluorescent Biosensors

    Type
    Blog Post
    ...Method Notes Quantification of a process Plate-based assays (plate reader) Detect the average amount of ...of fluorescence across a population of cells (i.e. in a plate well). Use to track a process (e.g. enzyme...often used to monitor enzymatic activity or phosphorylation over time. You can also use biosensors to follow...transcriptional activation, conformational changes, translation, and protein translocation. Targeting your biosensor...fluorescent HPLC (proteins or other molecules) Isolate or quantify cells, proteins or other molecules ...

  3. New CRISPR Web Resources and #12DaysofCRISPR Recap

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    Blog Post
    ...including histone acetylation by p300, histone demethylation by LSD1, cytosine methylation by DNMT3A or MQ1...HypaCas9 Day 10: Transcriptional regulation: Truncated gRNAs for Regulating Gene Expression and CRISPR 101...notice! Our newest page in the Transcriptional Regulation category already has five blue flame plasmids... MQ1, and cytosine demethylation by Tet1. RNA Targeting: This page contains Cas13 constructs for mammalian...

  4. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...completely digest the template DNA. Note—templates must have been isolated from methylation competent E. coli...and digest with methylation sensitive restriction enzyme DpnI to remove the templates. Digestion products...University of Connecticut. Molecular cloning is the isolation, insertion and amplification of a recombinant ...

  5. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    ...cells that will be labeled in a given population is directly correlated to the length of the truncated attP...subset of cells. By labeling fewer cells in a population, it’s easier to visualize individual/non-overlapping...transgenic animals are commonly used to specifically manipulate a cell type of interest in an organism, all cells...strategy results in sparse labeling of the cell population that expresses SPARC and the fraction of cells...intermediate (I), or sparse (S) labeling of the cell population. Figure 2: Dense (c), intermediate (d),...Clandinin TR (2019) SPARC: a method to genetically manipulate precise proportions of cells. bioRxiv 788679;...

  6. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    ...activity of a microtubule-associated protein to regulate cell growth dynamics Article contributed by Klaus... modified by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics with light by modifying EB1.... light - a finding which opens novel ways in manipulating and studying cellular dynamics. The photoinducible... faster than any genetic or pharmacological manipulations on cells, and has potential use for a wide range...Biology 2018 Pubmed PMID 29379139. The “Riboglow” platform for visualizing RNA production and localization...cobalamin binding riboswitches used in the Riboglow platform at Addgene. Braselmann E, et al. Nature Chemical...a new imaging-based bacterial colony screening platform, Oliver Griesbeck’s lab set out to engineer a ...

  7. What the HEK?

    Type
    Blog Post
    ...on all things HEK, from the first experiments isolating the cell line all the way to modern applications...applications. What the HEK is it?   HEK cells were first isolated in the 1970s by Alex van der Eb in the Netherlands...postdoc Frank Graham’s 293rd experiment, hence the nomenclature we use today: HEK-293. The exact origin of the... the fetal tissue which HEKs were isolated from is still a mystery, but it’s currently assumed that the...-day cell line is far removed from the initial isolate, it is difficult to determine its true tissue origin... to faithfully produce these proteins and its relatively low expression of native channels.  No matter...0995fje Tan, E., et al., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors...

  8. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ...subset of cells. By labeling fewer cells in a population, it’s easier to visualize individual/non-overlapping...this were labor-intensive and required using manipulations such as injection of precise titrations of AAV... of Ca2+ are high in the cell, calcineurin dephosphorylates the protein CZI, which in turn binds to calcineurin-dependent...calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target calcineurin...activity measurements in stressed or aging cell populations. Figure 3: Micrographs of wild type (...al., 2019. Looking for an alternative to intercalating fluorescent dyes for DNA-protein interaction...tTALE-eGFP and tTALE-mCherry. In comparison to intercalating fluorescent dyes which can cause photo-induced...

  9. Fluorescent Biosensors for Measuring Autophagic Flux

    Type
    Blog Post
    ...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3, that’s ...the cytosol, this ratio is low; but when Keima accumulates in autolysosomes, this ratio is high. When untargeted...biosensor contains mRFP and GFP fused to the autophagy-related microtubule-associated proteins 1A/1B light chain...quenched in acidic environments, while mRFP is relatively stable and fluorescences even at the acidic pH...mRFP-GFP-LC3 Tandem Fluorescent Protein, but following translation, this fusion protein is cleaved to generate one... GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...autophagic flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...

  10. Natural or synthetic? How Addgene’s dataset reveals trends in biological innovation

    Type
    Blog Post
    ...slight or large variations depending on how closely related these species are. If a gene was moved exactly ...exactly as is from one organism to a distantly related organism, the gene may not express well because of these...reference sequence database. Through a series of simulations and machine learning, they created a classifier...instances of rare codons, sometimes intentionally modulating expression in ways we don’t quite understand ...saw that a percent sequence identity below 85% correlates with synthetic sequences. The researchers then...growing trend of sourcing genes from distantly related organisms. In 2006, less than 1% of the genes deposited...deposited at Addgene were synthetic. But nine years later, over 20% of the genes deposited that year were ...

  11. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...for transcriptional regulation, so it requires a different approach for regulating transgene expression... generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail...systems currently lack the safety required for translational use. Viral Vectors Retroviral: Some of the...silencing of reprogramming transgenes and the up-regulation of corresponding endogenous pluripotency genes...Sano et al to learn how microRNAs can be used to regulate Sendai virus transgene expression. Transient .... The advantages of this method are that it’s relatively simple to implement and doesn’t require time-...the plasmid or silencing of the plasmid by DNA methylation. Another downside of using EBNA1 based vectors...

  12. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ...repressible promoters regulated? Like inducible promoters, repressible promoters can be regulated via positive ... the strong yeast promoter pTEF). As ethanol accumulates, it binds to the repressor, enabling it to bind...promoter pADH1m, which maintains activity during the late ethanol consumption phase of yeast culture. Repressible...One common binary system is the GAL4/UAS system isolated from yeast. In this system, UAS basal promoter...respective UAS and lexAop sequences but are also regulated by QS and quinic acid. From Riabinia et. al. Using.... et al. The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol ... and expanded Q-system reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed ...

  13. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...to do: gene knockout, a specific base edit, or modulation of gene expression. Location and sequence are... into DNA. This also requires an exogenous DNA template. HDR, however, is a very low-efficiency process... is >30nt from the proximal ends of the repair template (Yang et al., 2013). This means that, for gene...the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander edits are possible if...inhibition by CRISPRa and CRISPRi Finally, for modulating gene expression at the level of transcription...possible when using knockout or transcriptional modulation approaches. Avoiding off-target effects The ...genetic screens, however, a library composed of the latter would likely be more effective, so long as care...

  14. The Power Behind NGS Plasmid Validation: seqWell

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    Blog Post
    ...DNA from each well of a 96-well plate. Then, samples from several plates are pooled together to create ...sequencing to validate these constructs, as the relatively low cost and quick turn-around time of Sanger... sequential “pooled library prep”, in which a relatively large number of samples are subjected to a DNA-barcoding...samples individually, plexWell™ reduces every 96 well plate to a single tube after the first tagging step. As... samples (e.g. a collection of 96 plasmids on a plate) will typically have a wide range of input quantities... from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped to seqWell, where...

  15. The Crispr Babies Saga Shows the Need for Action, Not More Delays

    Type
    Blog Post
    ...attempt to create gene-edited babies. Several hours later, the scientist, Jiankiu He, confirmed that two such...editing techniques that are sufficiently safe to manipulate an embryo—even if it’s not necessarily via CRISPR...mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental groups...three branches – a private funder, government regulators, and an independent ethics council – all worked... process of good research practice: computer simulations, in vitro research, animal models, and several...question we are left with is not “what is the translational pathway for CRISPR/Cas9 research” but whether...stop punting decisions on scientific, ethical, regulatory, and general public consensus to the next meeting...

  16. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    ...consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses are varied... chromophore activates the response regulator via phosphorylation, promoting transcription of a GFP reporter...2005, when Tabor was working in Voigt's lab. Years later, these classic systems continue to be optimized ...the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription (see figure below)...spread across three plasmids, with the response regulator encoded in the genome. To make the system less...

  17. GCE4All: Making Genetic Code Expansion Accessible

    Type
    Blog Post
    ... an unnatural amino acid in translation (Noren et al., 1989). In the late nineties, this was developed...factors can negatively impact both translation and fidelity of translation, leading to system-wide effects...which can help determine structure and function relationships or, in more practical applications, allow a ...non-interacting with) the cells’ own natural translation machinery; these are often incorporated into ... any of a huge variety of ncAAs to study the relationship between structure and function…if you can get...

  18. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...glycoproteins and other proteins that require post-translational modification may not be easily expressed in ... E. coli and S. cerevisiae expression vectors in late March. The first set of 383 constructs went online...control processes. For the many other COVID-19 related plasmids in our collection, we’ve relied on Sanger...first pass QC. The lab team manually transforms, innoculates, and performs DNA minipreps for small batches...scientists prepare Sanger reactions by mixing this isolated plasmid DNA with appropriate sequencing primers...mutation.  Once the samples arrive, each group of plates will have select samples prepared and screened ...intensive screening and the use of high throughput platforms, Addgene and Ginkgo are striking a balance by ...

  19. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...PCR amplify the template plasmid. The parental, nonmutated DNA is digested with methylated DNA-specific ... a given disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged...DNA-specific enzyme DpnI, and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair...repair. This method can work well, but poor template digestion can result in a high number of background...mutations, to amplify both strands of a plasmid template. In addition to containing the mutations, the ...After being treated with DpnI to digest away the template plasmid, these PCR products are directly transformed...

  20. Antibodies 101: Validation

    Type
    Blog Post
    ...despite the manipulation. Additionally, not all sample types are amenable to genetic manipulation, nor can...your target then you would expect to see good correlation between the levels of target detected in both...both methods. These approaches have relatively low sensitivity, which can be problematic if you are trying...need to be aware of any possible alternative translation start sites or alternative splicing that could...also influence antibody binding by altering the relative concentration of potential antibody binders and... targeting a different epitope, and look for correlation between the results. Using antibodies against...cytometry using both antibodies, as well as a third unrelated antibody as a negative control (A). The two antibodies...

  21. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ...particles or relatively short expression times, where as others, such as hSynapsin, are relatively weak and...sites and the open reading frame is inverted in relation to the promoter, hence the name, double-floxed...homologous pairs, and that the degree of homology is correlated with the level of spontaneous recombination. ... etc.) and even neuronal phenotype in a mixed population (Burger, C. 2004, Aschauer, DF. 2013, O’Carroll...differences in the expression of receptors, post-translational modifications, and other factors that AAV utilizes...this study were packaged at Addgene and had a relatively low recombination rate (0.1% and 0.13% respectively... Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated...

  22. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... target required physical isolation of the visually identified subpopulation. For traditional cell culture...nucleotides of double-stranded RNA (dsRNA) results in downregulation, or “silencing,” of any gene that contains ... through targeted mRNA degradation, blocking translation, or chromatin modifications of complementary ...hairpin RNA, or artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent method...silencing efficiency and labor-intensive processes to isolate RNAi plants. With a visual reporter of RNAi, phenotypes...culture, enriching your active RNAi subpopulation can be easily achieved through fluorescence-activated...culture can be harvested instead of microscopic isolation. The plasmids required to conduct an APT-based...

  23. Finding Your Science Policy Path

    Type
    Blog Post
    ... diplomacy and international relations, advocacy, and writing legislation (see Figure 1 for career map... give advice and guidance to those that ask. Relationship building is key in science policy. Luckily, ...happy hours that you can attend to help build relationships.  Reach out to a science policy professional...can manifest via research and data gathering, regulatory efforts, policy implementation, program management... It occurs in a government setting: drafting legislation, making rules, and implementing the policy within...company who decides how they are going pursue regulatory approval for a new product or a well-endowed ...But some are also open to individuals from mid- to late-career backgrounds. In fact, many of my AAAS STPF...

  24. Plasmids 101: Modular Cloning

    Type
    Blog Post
    ... cellular behaviors requires making complex manipulations to cells. For example: in a cell there are proteins...other proteins and with DNA, and these complexes regulate the transcription of specific genes. What if one... one of those genes coded for a protein that regulated its own transcription, or the transcription of ...These interactions yield a cellular phenotype, translational output, etc., which can support a larger cellular... is made, modified (sometimes many times), and isolated afterwards. To engineer these production pathways... them to these organisms via another optimized platform. Gene Stacking As the name implies, gene stacking...identify the best cocktail of genes when the relationship between genotype and phenotype is best described...

  25. Plasmids for Endogenous Gene Tagging in Human Cells

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    Blog Post
    ...specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9...The coding sequence for GFP is transcribed and translated along with the gene, producing both a tagged ...the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, we used ...human genes and will add more to the collection later this year. Designing the donor plasmid Figure... one thousand base pairs on each side, in fact. Later, when we transfect the cells, these large regions...percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with...using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag ...

  26. Adenoviral Vector Production and Troubleshooting

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    Blog Post
    ... and then stained 10 days later. Plaques are counted and titer is calculated in terms of plaque forming...medicine (2017).   1st Gen AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot...8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response against AdV. Importantly...low titer rAdV-S, and can be stored at -80C for later use or used immediately for amplification.*Pro-Tip...not use CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count...260nm corresponds to 1.1 x 1012 particles/mL. To calculate the particle number: OD260 reading x dilution ...

  27. Rabies and Neuronal Tracing

    Type
    Blog Post
    ...the jobs they perform. This became foundational later on when we studied biological processes, like how...selectively infect a genetically defined cell population. While the EnvA genetic sequences are supplied...complex control of specifically defined neuronal populations. In one study, RABV monosynaptic tracing was ...neurons came from nearby neurons. It wasn’t until later time points that longer-range connections were seen...identify inputs to two major dopaminergic cell populations in two specific brain regions (Watabe-Uchida ...differences in the inputs to each of the two populations could be used to explain the differences in the... the functions of these two neuronal populations. Overall, this study laid the foundation for how different...

  28. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    Blog Post
    ...activity or their translation disrupted through physical blocking of the translation machinery. In the...gene expression and functions of key regeneration-related genes in four inexpensive and commercially available...transcription to create dsRNA. Students can produce DNA templates via PCR off of the same plasmid and use these ...expression changes through time or after genetic manipulation. The technique involves using synthetically ...create synthetic riboprobes off of linearized DNA templates which they can easily generate off of a circular... was first discovered and characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching...

  29. Design Tips for Prime Editing

    Type
    Blog Post
    ...reverse transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more...to the genome and a reverse transcriptase (RT) template including the desired edit. Figure 1: Prime...primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. The first...extension of the pegRNA should not be C. A C base is speculated to base pair with G81 of the gRNA (Anzalone et...encourage the cell to use the edited strand as a template during DNA repair. Test multiple nick sites, starting...R. (2021). Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell...

  30. Pooled Library Amplifications

    Type
    Blog Post
    ...liquid media or solid media (on plates). In general, amplification on plates can help to ensure that diversity... anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the library, but generally needs...transformation efficiency, or more specifically calculated based on the size of and weight of your library...library varies significantly in plasmid size). On plates, each transformed bacteria will be its own colony...Screening Zhang Lab - Multiplexed Overexpression of Regulatory Factors (MORF) Library 3,548 250 - 10,000...Screening Rinehart Lab Iterative Synthetically Phosphorylated Isomers (iSPI) Libraries ~11,000–110,139 ...

  31. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner...of auxin to the culture medium. Any number of regulatory sequences, including protein enhancers and repressors...repressors, can be included in the repair template for inducible alteration of protein expression levels...

  32. Quick Guide to Working with Drosophila Part 1: Getting Started with Flies

    Type
    Blog Post
    ...exciting directions. Drosophila are very easy to manipulate genetically and have limited genetic redundancy...faster but are also more stressed by the heat. Isolating male and female flies Differentiating male and...make genetic crosses properly, it is important to isolate females from males while they are still virgins...genomes Compared to humans and mice, flies have a relatively simple genome. They have four chromosomes: the...are miniscule, so researchers generally do not manipulate them. Essentially, the X, second, and third chromosomes... phenotypes, check out the Classical Genetics Simulator website. One interesting thing to know is that... systems. Some resources will direct you to the latest information about gene function, while others offer...

  33. Plasmids 101: Knockout/Knock-In Plasmids

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    Blog Post
    ...homologous recombination using a repair template. These templates can include loxP sites to create conditional...recombination. The concept is simple: deliver a template with a modified version of the targeted sequence...sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe the ... one of the homology arms. When we select cells later, we will first perform a positive selection for ...selection for the negative selection marker - this latter step will kill off many of the cells that have ...knockout targeting vector designed to insert and later remove a resistance gene. In this construct, the...

  34. Hot Plasmids and Viral Preps - May 2021

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    Blog Post
    ...VP1, which self-assembles into a compartment encapsulating the protein of interest (Figure 3).   ...D-glucaric acid biosynthesis. This MPyV system encapsulated a rate-limiting enzyme in this pathway (myo-... of the protein compartment. Yeast with this encapsulated enzyme produced 20% more D-glucaric acid, suggesting...system can improve metabolic pathway yields by encapsulating a single enzyme. Compared with previous research...captures rare clones that represent 0.001% of the population.  Because indel generation by Cas9 can occur ...steady rate, scientists found a way to use the accumulation of indels to measure time. These plasmids with...ready-to-use viral preps with more on the way. S5E2 regulatory element containing vectors developed in the Jordane...

  35. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...that grew on a selective plate versus the number that grew on a non-selective plate. Most bacteria tested... types of bacteria: lab-adapted, environmental isolated, and disease-associated. Mobile-CRISPRi: a modular...glmS gene in the recipient's genome. Antibiotic plates lacking DAP select against donor bacteria and for...including pathogenic strains and environmental isolates. For example, Peters and colleagues used Mobile-CRISPRi...aeruginosa) in addition to Vicrio casei, a bacteria isolated from cheese. Knockdown efficiency, as measured... 100-fold excess of wild-type bacteria and the relative frequency of each of the 40 library strains was...

  36. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    ...screening experiment is to generate and screen a population of mutant cells to identify genes involved in...experiments commonly use lentivirus to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral...CRISPR libraries”) consist of a heterogeneous population of gRNA-containing lentiviral transfer vectors...the CRISPR library to generate a heterogeneous population of mutant cells, with each cell or set of cells...distribution is analyzed in the drug-resistant population compared to a non-treated control group. In this...maintaining the distribution of each gRNA within the population is key. Loss of representation due to enrichment... sufficient representation within your mutant population requires a massive amount of cells as starting...

  37. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    ... for screening protein-protein interactions. In later years, improvements to yeast transformation protocols...can either be applied to the bait in a single population (e.g. transforming both fusion plasmids, or mating... strain for screening), or arrayed on a 96 well plate where each well contains a defined prey fusion.  ...former allows for screening greater numbers, but the latter eliminates the step of identifying prey proteins...protein expression and correct folding, post-translational modification, protein degradation, access to...problem is that your proteins are not post-translationally modified, co-expression of the enzyme responsible...transcription-activating function of a eukaryotic regulatory protein. Science. 1986; 231:699-704. PubMed PMID...

  38. Performing In Vivo CRISPR Screens Using the FITS Approach

    Type
    Blog Post
    ...in immune cells have identified many putative regulators of immunity. However, in vitro screens cannot... a Thy1.1 selectable marker to facilitate the isolation of modified cells; and (5) is compatible with ...conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses to a murine...responses to cancer. The screen recovered known regulators, such as Ptpn2, Pdcd1, and Zc3h12a, demonstrating...Moreover, the screen identified putative novel regulators of CD8+ T cell responses in tumors, demonstrating...Cancer and the Immune System: Deciphering the Relationship...

  39. Viral Production at Addgene

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    Blog Post
    ...Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation. Sequencing results...of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins...SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed...Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. ...are added to cells in culture. Three to five days later, cell cultures are inspected for sterility. Transducibility...-AAVrg) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with ...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...

  40. CRISPR 101: Targeting RNA with Cas13a (C2c2)

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    Blog Post
    ...dCas13a may be useful for isolating specific RNA sequences from a population (either enriching or depleting... proteins for imaging, tracking, modulating splicing, and regulating expression of specifically targeted...Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but they require both a tracrRNA and crRNA...the spread of infection throughout the entire population. This property of Cas13a opens up the possibility...knockdown to single gRNA expression. Although the collateral RNA cleavage response is well described in bacterial...
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