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We narrowed to 162 results for: Mammalian expression plasmids

Showing: 121 - 140 of 162 results

  1. Fluorescent Proteins 101: Fluorescent Protein Timers

    Type
    Blog Post
    Published
    May 4, 2017, 2:30 p.m.
    ...fluorescent protein timers When FP timers are first expressed, they predominantly fluoresce one color but slowly...timers (whether as part of protein fusions or expressed independently) originally fluoresce green or blue...displays predictable kinetics over 14 hours of expression in C. elegans embryos. However, this initial ...ideal FP timers: 1. Ratiometric determination of expression time The ratio of mature to immature fluorescence...fluorescence from the FP timer is dependent on the total expression time but independent of protein concentration...Calibration curves correlating this ratio to the total expression time can therefore be used to determine how long... long a pool of FP timers has been expressed, either in whole cells or as a pool of fusion proteins localized...

  2. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...this evolving field! Find prime editing plasmids! Find plasmids from David Liu's lab! This post was originally...the CRISPR guide Find CRISPR plasmids by function Browse our entire plasmid collection ...: Optimizing prime editor sequence to improve expression and activity In addition to the numerous innovations... the RNA pseudoknot sometimes altered epegRNA expression compared to pegRNAs, prime editing efficiency...efficiency was improved even in contexts where expression was reduced (Nelson et al., 2022). PE7: An alternate...identified a protein that enhances prime editing in mammalian cells: the small RNA-binding exonuclease protection...La. They found that La, which is ubiquitously expressed in eukaryotes, binds and stabilizes the 3′ tail...

  3. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ... tag plasmids here! Where should I place the tag and how many do I need? When designing a plasmid containing...tag is a string of 6 or more histidine residues expressed in frame on the N- or C-terminus of a recombinant...histidine-containing protein contamination, such as mammalian production systems, consider using these more ...ion substrate. If your protein of interest is expressed at a very low level, try a longer histidine tag...from Addgene More Resources on the Addgene blog Plasmids 101: Protein Tags Antibodies 101: ChIP Antibodies...purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad ...

  4. An Introduction to Adenovirus

    Type
    Blog Post
    Published
    Oct. 3, 2023, 1:15 p.m.
    ...commonly derived from the mastadenovirus members (mammalian-infecting members) of the Adenoviridae family,...reconfiguring of the nuclear compartment, gene expression, and protein translation. Its genome consists...vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early 3) genes .... These genes’ functions are rescued by helper plasmids used during production and/or by genes present...made available by this deletion from the genomic plasmid can be used to house a recombinant sequence (transgene...late genes are usually present in the transfer plasmid for the majority of adenoviral vector systems used... used in research, they are only expressed in the producer cells where there is E1 and E3 gene complementation...

  5. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    Published
    June 14, 2018, 11:44 a.m.
    ...death of transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of ...their own but can be produced in E1-containing mammalian cell lines such as HEK293 cells. 1st generation...capacity is limited to 8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response... capacity (up to 36 kb!), long-term transgene expression and negligible toxicity. However, their production...down into 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular...adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene of interest...how to generate the final recombinant adenoviral plasmid construct, please see Addgene’s Adenoviral Guide...

  6. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...background via P1 transduction. Plasmid Expressing lambda red genes from a plasmid allows for a mobile recombineering...cI857) to limit leaky expression of the Red system.  Using a plasmid to express the lambda recombineering...activation Expression of Lambda Red recombination genes There are four ways to express the lambda red...Controlling expression of Red proteins is critical for minimizing the toxic effects of Gam expression and to...lambda red expression is that it doesn’t require antibiotic selection to maintain expression of the recombineering...promoter.  It is a good idea to choose plasmids that also express the repressors associated with these ...  A simple way to do this is to express lambda red genes from a plasmid with a heat sensitive origin of...

  7. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    Published
    Dec. 16, 2015, 3:30 p.m.
    ...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking... modified for genome editing, activating gene expression, visualizing genomic loci, and much more. Now...included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting...compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene here! Future possibilities...should enable researchers to make precise edits in mammalian cells and may decrease worries about off target... (2015): aad5227. PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu..." Nature (2016).  PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS...

  8. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    Published
    Feb. 28, 2017, 3:30 p.m.
    ...gRNA was present or b. when a gRNA binding a non-mammalian sequence (GAL4) was supplied. In addition to ...hairpins generates white light. All 6 gRNAs can be expressed in a single vector, pCRISPRainbow-DONOR1, with...CRISPRainbow gRNA will permit labeling, but a newly expressed standard length gRNA would “switch” the system...24360272. PubMed Central PMCID: PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui...25713381. PubMed Central PMCID: PMC4364232. Find the plasmids from this publication at Addgene. 3. Ma, Hanhui...27088723. PubMed Central PMCID: PMC4864854. Find the plasmids from this publication at Addgene. 4. Kiani, Samira...26344044. PubMed Central PMCID: PMC4666719. Find plasmids from this publication at Addgene. Additional ...

  9. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    Published
    April 12, 2016, 2:30 p.m.
    ...pool of lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid libraries containing...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...and ligation steps to build barcoded lentiviral plasmids containing one or more gRNAs. As each ligation...containing combinations of different gRNAs in each plasmid. Each gRNA combination can be tracked and quantitatively.... "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library...Background with Our CRISPR Guide Pages Find CRISPR Plasmids Find More Tools for Combinatorial Engineering ...

  10. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...genome and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes...crossover event between the vector plasmid and the packaging plasmid caused the Psi sequence to be placed... facilitates nuclear export) is expressed from a separate plasmid, and the promoter of the 5’LTR has been...(tTA, also expressed by your transfer vector) binds to the TRE and activates the expression of your gene...usually 293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...development of viral vectors, gene transfer into mammalian cells often relied on chemical (CaCl2, cationic...

  11. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...magnetoresponse was clearly visible whether expressed in E. coli, in mammalian cells, or in purified form, and didn...resources on addgene.org Find Optogenetics Plasmids Find Biosensor Plasmids Visit the Fluorescent Protein Guide...Frank Hayward et al., 2024). They deposited their plasmid with Addgene, hoping to inspire others to explore... the very first protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable...one-component magnetoresponsive system that could be expressed and purified. Although a ~1% change in signal ...   Video 1: Fluorescence movie of E. coli expressing MagLOV while a magnet is waved under the sample...Lab is eager to hear from you. Find the MagLOV plasmid here!   References and resources References Duret...

  12. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ... The ready-made PITCh plasmids available from Addgene are perfect for expressing GFP from a given promoter...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...cloning. An overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and ...sites. This PITCh plasmic is cotransfected with a plasmid carrying Cas9, the PITCh-gRNA, and the locus-specifc.... The PITCh-gRNA creates two DSBs in the PITCh plasmid to release the cassette, and an insertion-locus-specific...

  13. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

    Type
    Blog Post
    Published
    March 5, 2019, 1:08 p.m.
    ...promoter is expressed. Cre-mediated deletion of one or several of these genes permits the expression of genes...activate the expression of a different fluorescent protein. Therefore, each cell could express a unique combination...copy of the transgene expressing either YFP or CFP, and cells that do not express Cre fluorescing with ...the low expression of XFP of Brainbow by incorporating a Tet-Off system to boost XFP expression. The increased...variety of colors when a combination of XFP are expressed. Each cell remains the same color for its entire...of the downstream OFP, GFP, or RFP. Cells that express the construct but do not undergo recombination ...truncated YFP. In Thy1-Brainbow mice, some neurons expressed as many as 16 copies of the injected transgene...

  14. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    Published
    June 25, 2019, 12:54 p.m.
    ...don't exactly "eat" plasmids as depicted in this comic, when a bacterium uptakes a plasmid, the genes on the... the plasmid can give them different phenotypes. For example, this plasmid encodes a green fluorescent... Read all of our Plasmids 101 blog posts Check out all of our blog posts on plasmid cloning Resources...comes from adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment...membrane so the bacteria can easily uptake your plasmid of interest. Scientists can also use electroporation...exist as transient extrachromosomal DNA, like a plasmid, or it can integrate into the host bacterium’s ...cloning vector that contains both bacteriophage and plasmid properties. The phagemids are packaged into replication-incompetent...

  15. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    Published
    July 12, 2016, 2:30 p.m.
    ...Resources on the Addgene Blog CRISPR 101: Mammalian Expression Systems and Delivery Methods Learn about... Find CRISPR Plasmids for use in Mammals Check out Our CRISPR Guide Pages Find Plasmids for Mouse Targeting...with, making them the go-to choice for studying mammalian biology. Similar to any model, mice are not without...the target site, and a donor oligonucleotide or plasmid as the repair material if needed (for knock in ..., the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together and microinjected...provided (in the form of an oligonucleotide or plasmid), precise changes can be made in the genome via...mice carrying the intended mutation. With a donor plasmid, the outcome is less predictable. In our best case...

  16. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    Published
    May 20, 2014, 2:06 p.m.
    ...of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to ... The two most common uses are: 1) to test the expression level in a specific system (by measuring the ...optimized for 37°C, and is therefore most suited for mammalian or bacteria studies, whereas GFPS65T is better... parameter is important if you are planning to express the FP in acidic environments (e.g. yeast cytosol...He is interested in everything related to gene expression, particularly at the RNA level. He maintains ... comprehensive data sheets on many FP variants Plasmids 101: Green Fluorescent Protein (GFP) Three Review...proteins and imaging technologies to study gene expression, nuclear localization, and dynamics” Curr. Opin...

  17. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ... as a donor plasmid containing the transposon, along with a helper plasmid that expresses the transposase...lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...the dose of plasmid the cells receive over the whole reprogramming period and the plasmid will get diluted...larger plasmids have lower rates of transfection. Minicircles: Minicircles are like mini-plasmids. They...

  18. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    Published
    May 31, 2016, 3:30 p.m.
    ...the ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source... minimize expression levels of your gene of interest, as needed. Controlling the expression levels of ...useful resource. While Addgene cannot distribute plasmids with the piggyBac™ transposase itself, please ...successfully used for genetic manipulation in mammalian cells until 2005 [1]. Like other transposons, ...genome fully intact, while, with a standard DNA plasmid transfection, a double-stranded break may occur...may be particularly useful when the gene being expressed has threshold effects, such as with signaling ... can also mitigate the deleterious effects of expression of certain genes, such as those that have negative...

  19. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    Published
    Oct. 30, 2018, 1:04 p.m.
    ... Visit Our Stem Cell Page Find Plasmids for Reprogramming Find Plasmids for Differentiation ...the embryonic and extraembryonic cell types. In mammalian development, cells found in the zygote through...reprogramming differentiated adult cells by the ectopic expression of four embryonic genes- OCT4, SOX2, KLF4, and...to a Nobel Prize for Dr. Yamanaka in 2012. Find plasmids for generating iPSCs iPSCs and ESCs closely resemble...possess similar morphologies, growth properties, and express stem cell markers similar to ESCs [11].  While ...

  20. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    Published
    April 9, 2019, 1:13 p.m.
    ...native expression levels. However, it is advisable to first express the fusion protein from a plasmid-based...instance, Lifeact-mTurquoise2 (Plasmid #36201) or mScarlet-tubulin (Plasmid #85045). We often use restriction...fluorescent proteins that behave as monomers in mammalian cells include: mTurquoise2, mEGFP, mNeonGreen,...strategy (Sheridan, 2002). Avoiding protein over-expression artifacts The introduction of DNA that encodes...of protein within a cell and may lead to over-expression artifacts. This is an important downside of the...Download the fluorescent proteins 101 eBook Read our plasmids 101 blog series Resources on Addgene.org Browse...protein fusions Browse the fluorescent proteins plasmid kits ...
Showing: 121 - 140 of 162 results