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We narrowed to 370 results for: Cre expression vector

Showing: 81 - 120 of 370 results

  1. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ...elements for vector propagation. This post, along with a future companion post on mammalian vectors, will catch...typically found as a single copy. Unlike the ARS vectors, CEN vectors are stable without integration. Yeast Episomal...of marker gene expression is to use a partially defective promoter to drive expression of the selection...to be created in advance of the experiment. The marker provided by the plasmid may be expressed at higher...plasmid maintenance within an E. coli cell, but vectors can be widely utilized across many different cell...core replication and resistance features of yeast vectors and explain how they differ from the bacterial ...previously described. Why do scientists use yeast vectors? Yeast are eukaryotes and thus contain complex ...

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

    Type
    Blog Post
    ...cerevisiae expression vectors are available at Addgene. A set of mammalian expression vectors containing... be easily expressed in non-mammalian cells. Yeast expression plasmids The yeast expression constructs....  Bacterial expression plasmids For those constructs to be used in bacterial expression systems, Ginkgo...backbones which drive expression using either TEF1 or GAL1 promoters. These vectors are intended for the...tag, cleavage, codon usage, and expression system. Mammalian expression plasmids Finally a set of wild ... (spike protein) cloned into a CMV driven expression vector are also available. These contain no tags ...coding genes in E. coli and S. cerevisiae expression vectors in late March. The first set of 383 constructs...

  3. Plasmids 101: E. coli Strains for Protein Expression

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    Blog Post
    ...to reduce basal expression levels; expression vector cannot have p15A origin of replication F- ompT lon...) pLysE has higher T7 lysozyme expression than pLysS; expression vector cannot have p15A origin of replication...Rhamnose-tunable T7 RNAP expression alleviates inclusion body formation. Expression vector cannot have p15A origin...for recombinant protein expression. Many challenges can arise when over-expressing a foreign protein in ...to avoid them. Why do I need an expression strain? Protein expression from high-copy number plasmids and...Addgene's curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains ...General protein expression BL21 (DE3) pLysS* Chloramphenicol (pLysS) pLysS expresses T7 lysozyme to ...

  4. New and Upcoming Viral Vectors - December 2019

    Type
    Blog Post
    ...pAAV.hSynapsin.SF-iGluSnFR.A184S Viral vectors coming soon! These vectors should be packaged and available ... of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...new tools to our inventory of ready-to-use viral vectors. Here are some of the AAV we have released in the...pAAV-hSyn Con/Fon hChR2(H134R)-EYFP that is both Cre and Flp dependent. Plasmid Serotype Name 124603...only interpret their results, but also confirm expression in target cells. While the various AAV serotypes... can be used to achieve different patterns of expression, researchers often need to determine these transduction...under control of the CAG promoter for ubiquitous expression. (For more details on this technology, see our...

  5. Tips for Screening with Yeast Two Hybrid Systems

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    Blog Post
    ...Yeast Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About Yeast Vectors Read...activation. Vary the expression levels of bait and prey proteins - overexpression can lead to false interactions...interactions. Lowered expression levels will increase the stringency of your binding. And most importantly...ends’ of your protein are screened for binding. Using a variety of expression levels and systems can alleviate...produce a more vigourous screen is to use a number of different bait and prey vectors. This has been shown...During this period, proteins were successfully expressed as fusions that retain their individual activities...see Figure 2a) (4). These fusions are then co-expressed in a yeast strain that also contains a reporter...

  6. Chromoproteins: Colorful Proteins For Molecular Biology Experiments

    Type
    Blog Post
    ...that have excised the vector backbone. Vectors pTOX4, pTOX5, and pTOX6 express the YhaV, MqsR, and Tse2...excision of the vector backbone. To do this, rhamnose is added which induces expression of the toxin. Any...encode the amilCP chromoprotein. Vectors pTOX7, pTOX8, and pTOX9 express the YhaV, MqsR, and Tse2 toxins...chromoproteins to create negative selection markers for allelic exchange. These vectors contain a toxin ...gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria on agar plate. Image: Liljeruhm...intensities, maturation times, and fitness cost of expression. They found that the cellular fitness cost varied...lost color overnight in liquid culture, though expressing the chromoproteins from the chromosome could ...

  7. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...selectable markers that are only expressed when the targeting vector is inserted downstream of a functional...them sequentially into the chosen acceptor vector. The vectors are then packaged into AAV virions via transient...Check out our Viral Service Find AAV Vectors Browse all Viral Vectors ...fantastic gene delivery vehicles for episomal gene expression and are particularly useful for gene delivery...well understood, researchers have been using AAV vectors for gene editing in mammalian cells since 1998....when delivered via AAV infection, gene targeting vectors with relatively short (1 kb) homology arms underwent...orders of magnitude higher than identical targeting vectors delivered through transfection of naked DNA. Other...

  8. Using Addgene's New Viral Service for Your Research

    Type
    Blog Post
    ...Expanding the viral vector knowledge base: You can help! We’ve been expanding our viral vector educational resources...Beginners Lentiviral Vector Uses and Overview AAV: A Versatile Tool for Gene Expression in Mammals Additional...gene, replace it, modulate its expression, or perform a genome-wide screen, we’ve got viruses to help you...spending time and money producing virus from select vectors in the repository yourself, you can now order ready-to-use... production process while still ordering viral vectors. Virus production can be a frustrating process ...Lentivirus As some of the most popular lentiviral vectors in the repository are used to produce CRISPR tools...experiments, while rtTA can be used to activate expression of a gene under the control of the tet operator...

  9. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...yeast expression, worm expression, insect expression, plant expression, mouse targeting, CRISPR, TALEN,...mammalian expression, bacterial expression, lentiviral, retroviral, AAV, RNAi, luciferase, cre/lox, yeast...biosensor affinity for calcium.”  Primary Vector Type The vector type should describe how your plasmid ...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids ...and Organization where your plasmids were first created. From there, we’ll use the information in the ...

  10. Hot Plasmids - August 2020

    Type
    Blog Post
    ...easier to clone, express, and purify compared to antibodies. Nanobodies are easily expressed in the cytosol...#140702) The pMAK461-64 plasmids were expressed in SHuffle T7 Express competent E.coli (NEB) using IPTG induction...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly ...fluorescently labeled piggyBac vectors for uniform and sustained expression of single or multiplexed gRNAs...and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector integrates into ...First, the authors created the pMAK backbone by modifying pMAL-T-Avi-His/BirA, a vector commonly used for... the mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image...

  11. Harnessing Bacterial Toxins for Allelic Exchange

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    Blog Post
    ...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an...Tight control of toxin expression is absolutely necessary, because leaky expression of toxin could select... interfering with expression of surrounding genes. However, there are ways to create scarless mutations...recombineering (Khetrapal et al., 2015). They created vectors for Lambda Red that contained one of eight ...introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette...selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover...leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...

  12. Plasmids 101: What is a plasmid?

    Type
    Blog Post
    ...Vital component for expression vectors: determines which cell types the gene is expressed in and amount of... referred to as “vectors” or “constructs.” To insert a gene of interest into a vector, scientists may ...plasmid with its elements described below.   Vector Element Description Origin of Replication (ORI...sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a ...Regardless, once the cloning steps are complete, the vector containing the newly inserted gene is transformed...Generally, scientists use plasmids to manipulate gene expression in target cells. Characteristics such as flexibility...common plasmid types include cloning plasmids, expression plasmids, gene knock-down plasmids, reporter ...

  13. Getting the Most from Your Lentiviral Transduction

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    Blog Post
    ...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...often employed to establish cell lines that stably express a gene of interest. While the general approach ...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...compete with host cells for nutrients and can alter expression of receptors, ion channels, and growth factors...on a variety of factors such as the transgene expressed, the viral storage buffers used, and other experimental... by the envelop protein. Many times lentiviral vectors are engineered with non-native envelopes, a process...will be able to overcome differences in receptor expression. It's all in the mix Even before the lentiviral...

  14. Viral Vectors 101: What is a Virus (and a Viral Vector)?

    Type
    Blog Post
    ...plasmids and expressed via cells cultured with the vectors. This allows the viral vectors to reproduce...Viral Vectors 101: Viral Applications Viral Vectors 101: Viruses as Biological Tools Viral Vectors 101:...able to create far safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses..., just to increase the viral vector’s carrying capacity. In order to allow the viral vectors to reproduce...of Viral Vectors 101 posts about each type, so be sure to check our blog, or our Viral Vectors 101 eBook...As a bonus, this increases the carrying capacity of the viral vector, increasing both safety and usefulness...removed. This means the vector can deliver a genetic payload to a cell but cannot create new particles. As ...

  15. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    ...Cas9. Adenoviral CRISPR vectors (left) or CRISPR vectors and donor DNA vectors (right) are transduced ...need to use a two viral vectors system: one vector for Cas9/sgRNA and one vector for your specific DNA ...gRNA and Cas9 using adenoviral vectors As compared to other viral vectors, AdVs can provide a large transgene...than AAV vectors. This capacity is large enough to carry the Cas9 gene and a gRNA expression cassette ...adenoviral vectors Since then, other groups have successfully developed and used AdV vectors to edit genes...to labs wishing to create this kind of animal models. Start using adenoviral vectors with your CRISPR/Cas9...updated Dec 10, 2020 by Benoit Giquel.   Adenoviral vectors (AdVs) have been used for many years for in vivo...

  16. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...be used to create recombinant vectors that can replicate in different bacteria and express proteins within... S. (2011). Broad-host-range plasmid vectors for gene expression in bacteria. Methods in Molecular Biology...Gill, R. T. (2010). Broad-host-range vectors for protein expression across gram negative hosts. Biotechnology...Cloning Vectors for Gram-Negative Bacteria. In R. L. Rodriguez & D. T. Denhardt (Eds.), Vectors (pp. 287...in nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...can harness those features to create broad host range recombinant vectors.  A plasmid’s host range is ...range replicons, recombinant vectors can be modified further to increase the range of hosts in which proteins...

  17. Adenoviral Vector Production and Troubleshooting

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    Blog Post
    ...method for creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...Three generations of adenoviral vectors: the first generation vectors are deficient in the E1 and E3 regions... second generation vectors are deficient in E1, E2, E3, and E4. Gutless Ad vectors (helper-dependent (...therapy due their increased cloning capacity (up to 36 kb!), long-term transgene expression and negligible...death of transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of ...Adenovirus type 5. Three generations of adenoviral vectors The adenovirus virion is a non-enveloped particle...

  18. CRISPR 101: Mammalian Expression Systems and Delivery Methods

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    Blog Post
    ...System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible...electroporation. Transient expression of CRISPR components, expression decreases as RNA is degraded within...cationic lipids. Transient expression of CRISPR components, expression decreases as gRNA and Cas9 protein...included with each expression system as well as suitable applications. Expression System Components ... in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene (e.g. ... AAV transfer vector and used to generate AAV particles. Transient or stable expression of SaCas9 and/...repress gene expression? Are you trying to create a single point mutation? Do you want to create a fusion ...

  19. Tips for Titering Your Lentiviral Preps

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    Blog Post
    ...target cells that are positive for vector encoded transgene expression following transduction with serial...target cell line with your virus and assaying for expression of a gene carried on the transfer plasmid or ...primers that target the common features of viral vector backbones; once validated these universal primers...higher than for functional assays depending on the vector backbone. When using qPCR based methods for viral...plasmid which can over-inflate results. A packing vector deficient in reverse transcriptase will produce... prep. This method is particularly useful when vectors carrying fluorescent markers are used as it eliminates...addition, promoter choice can also impact transgene expression. The most accurate method of lentiviral titration...

  20. Plasmids 101: The Promoter Region – Let's Go!

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    Blog Post
    ...Description Expression Additional considerations CMV General expression mRNA Strong mammalian expression promoter...lower expression than U6. May have better expression in neuronal cells. U6 small RNA expression shRNA...promoter with somewhat weak expression. lacIq mutation increases expression of the repressor 10x, thus ...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor ...give consistent expression regardless of cell type or physiology. SV40 General expression mRNA Mammalian...  Commonly used in expression systems for Drosophila. Polyhedrin General expression mRNA  Strong insect...Constitutive  Commonly used in expression systems for insect cells. CaMKIIa Gene expression for optogenetics mRNA...

  21. PiggyBac-ing Through the Genome Editing Field

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    Blog Post
    ... minimize expression levels of your gene of interest, as needed. Controlling the expression levels of ...quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences distributed throughout...encourage you to deposit your piggyBac™ transposon vectors with us to help us expand this useful resource....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...piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While this method...edit of interest. With the larger piggyBac™ donor vector, it is possible to make the ITR and selection cassette...

  22. 15 Hot Plasmids from 2017

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    Blog Post
    ...low off-target activity; mammalian expression vector Rees H, et al. Nature Communications. 2017. PubMed...and ribozyme systems for multiplex gRNA expression. Expression of toolkit components can be controlled...gene editing. The bacterial and mammalian expression vectors for these CRISPR inhibitors are listed in...Species Bacterial Expression Construct SpCas9 Inhibition (E. coli) Mammalian Expression Construct SpCas9...observed reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work ...TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that have standard cloning sites or gateway compatible...neuronal tools to the expanding gateway compatible vectors currently shared amongst the zebrafish community...

  23. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    Blog Post
    ...interference (RNAi) to knockdown expression of specific gene expression. The 49 deposited plasmids enable... all have a PR-T4 vector backbone, which is a modified version of L440. This vector is versatile in that...of interest into the empty PR-T4P vector backbone. The empty vector PLL4, which is similar to PR-T4P, ...Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians." Proceedings...Scientists routinely use techniques to alter gene expression or to label specific cells, but there are too... in Situ Hybridization (WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated...enable students to study gene expression and functions of key regeneration-related genes in four inexpensive...

  24. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

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    Blog Post
    ... cloned into an entry vector. The 2in1 cloning system contains two entry vectors (pUC57-L1L4 and pUC57... of protein coexpression. By introducing a single plasmid containing both GOIs, expression levels are ...-protein interaction studies easier with a 2in1 vector cloning system Article contributed by Michelle ... Recently, the Grefen lab introduced the “2in1” vector cloning system - a Gateway compatible approach ... genes-of-interest (GOI) into two independent expression cassettes on a single plasmid (Grefen and Blatt...digest cloning to introduce the GOIs into the 2in1 vector instead of the GatewayTM cloning BP reaction, where...Agrobacterium-mediated DNA transfer, each GOI is usually expressed from a different plasmid, which may lead to unequal...

  25. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

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    Blog Post
    ...multiplexed gRNA expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12 ...below and pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC...barcode, respectively. Once cloned into the storage vector, the pooled, barcoded gRNA library is now ready...pAWp12 lentiviral vector should retain the diversity in your gRNA library, but this should be verified by NGS...means to create barcoded gRNA libraries that can be used to combinatorially modify the genome, screen for ...revolutionized how we decode the genome by making it easy to create specific genetic perturbations. The ease with which...

  26. Protocol - Bacterial Transformation

    Type
    Protocol
    ...plasmids (even those designed for mammalian cell expression) carry both a bacterial origin of replication...approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. Incubate the...Scientists have made many genetic modifications to create bacterial strains that can be more easily transformed... specific treatments have been discovered that increase the transformation efficiency and make bacteria...

  27. Improving Transduction Efficiency with Exosome AAVs

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    Blog Post
    ...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector production...acids. This conjunction of vectors and exosome was given the name vexosomes (vector-exosomes), exosome AAVs...transfecting the producer cell line with an expression vector for CD9 (a known marker molecule for microvesicles... of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...production, assembled AAV vector particles accumulate inside the producer cells and purification protocols...purification steps. It’s also possible for AAV vectors to be spontaneously released from the producer ...Harvard Medical School took a closer look at the AAV vectors that are released from cells during production ...

  28. Save Time with Transient Plant Leaf Transformations

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    Blog Post
    ...observe expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the...us. Use a well working vector system This allows you to quickly assemble vectors and test different ideas...MoClo system. The vectors mentioned in this blog post above are ready to use vectors, but with the MoClo...reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables us ...MoClo system you can easily and quickly create customized vectors. For more information on MoClo for plants...reporter gene so that the transformation and gene expression can be tracked. During a transient leaf transformation...References D'Aoust, Marc-André, et al. "Transient expression of antibodies in plants using syringe agroinfiltration...

  29. Exploring Applications of the Bioluminescent HiBiT Tag

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    Blog Post
    ...quantifying variant expression with a minimally disruptive tag. Find HiBiT-TP53 Fusion Vector at Addgene! Following...HiBiT. When EVs from cells expressing ADAR1-HiBiT were added to LgBiT-expressing hepatocytes or co-cultured...low-levels of ectopic expression, thus avoiding artifacts common in overexpression models. Although HiBiT...signal, strong enough to detect from proteins expressed at their endogenous levels. HiBiT is compatible...cell models, and even introduce mutations. This vector-based use of HiBiT provides a means to quickly ...to provide complementary validation of protein expression and localization. HiBiT-tagged targets in translational...plasmids from the Promega Plasmid Collection that express similar constructs available at Addgene. Read the...

  30. Viral Vectors 101: Gamma-Retroviral Packaging Systems

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    Blog Post
    ...Addgene blog Viral Vector Elements The Retroviral Life Cycle Popular Retroviral Vectors and Their Use in...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...element; sequence that stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents...method utilizes a packaging cell line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver...  Frequently asked questions about γ-retroviral vectors How do engineered γ-retroviruses differ from lentiviruses...reduce promoter interference and/or drive high expression of a transgene from an internal promoter. An ...was originally published in 2018, in the Viral Vectors 101 eBook, 1st edition.  References and resources...

  31. The Importance of High Titer for AAV Transductions

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    Blog Post
    ...a roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...observable expression. Doubling this titer to 1012 resulted in expression in roughly 200 cells. Increase the ... produce expression. The higher the titer, the better. AAV transport and transgene expression It’s clear...infect a cell to achieve visible expression of the protein. However, expression of receptors may not need to...concentrate this type of vector to such a high titer when looking for effective gene expression. How do you achieve...to the nucleus where their gene cargoes can be expressed. The bottom line is that a lot of AAV particles...transduction. In another example, neuronal gene expression by an anterograde AAV was shown to drop off drastically...

  32. Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics

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    Blog Post
    ... of gene expression and reduces toxicity as compared to previous Cre/lox or drug-induced Cre systems (... Figure 4: The C-terminus of Cre (CreC) and the N-terminus of Cre (CreN) are fused to the truncated Phytochrome...As the name suggests, CreLite combines the Cre/lox system with optogenetics. Cre activity is controlled...determined protein expression levels from different promoters, and inhibited gene expression using CRISPRi.... dimerize, bringing each terminus of Cre together to restore Cre activity. As soon as red light or PCB...proteins separate, inactivating Cre. This is critical for reducing Cre toxicity in long-term experiments.... In this system, cells express various fluorescent proteins controlled by Cre recombination. Each cell...

  33. Viral Vectors 101: Parts of the AAV Packaging Plasmid

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    Blog Post
    ...drives Cap expression. Often Rep proteins from the AAV2 serotype are used to package vector genomes flanked...AAV (rAAV) is made in the lab, rep and cap are expressed from a separate plasmid in trans, freeing up space...packaging: Rep78, Rep68, Rep52, and Rep40. Rep gene expression is often driven by the AAV p5 or p19 promoters...Strohl WR (2017) Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31:317–334 . https:/...for Targeting and Improving the Efficacy of AAV Vectors. Molecular Therapy - Methods & Clinical Development...Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction. Human Gene Therapy...Figure 1. The RepCap plasmid and its products. Created with BioRender.com   Replication (rep) gene...

  34. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger expression cassettes ...chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within...the Rapid Design and Production of Diverse Expression Vectors.” ACS Synthetic Biology, vol. 6, no. 7, American...technology has been used to optimize protein secretion and expression in the biotech workhorse yeast — K. phaffii...GoldenBraid Modular Cloning System and Creation of a Toolkit for the Expression of Heterologous Proteins in Yeast... tools can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even...from individual transcription units (TUs) used to express your favorite gene-of-interest, all the way to ...

  35. A Primer on Optogenetics: Introduction and Opsin Delivery

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    Blog Post
    ...with an appropriate mouse line expressing a particular cre, viral expression of your opsin is no longer required...steps in the coming weeks. Expression of the optogenetic constructs Expression of your desired optogenetic...promoter-directed expression alone is extremely limited. Therefore, highly expressing, generic neuronal...promoter of the cre recombinase while still taking advantage of the robust expression via the promoter...Finally, expression of the opsin in a mouse line will likely be less than that of viral expression but may...stimulation. Testing opsin expression  Regardless of the method of expression of your opsin gene, it is...extremely high expressing promoters). Conduct a time course experiment in which you monitor expression at various...

  36. Performing In Vivo CRISPR Screens Using the FITS Approach

    Type
    Blog Post
    ...feature of this system is a lentiviral gRNA expression vector (pRDA_526) that (1) enables efficient knockout...murine tumor model in vivo. In this screen, we used the pRDA_526 vector to knock out 30 genes putatively ...the system and utility of the pRDA_526 vector. Moreover, the screen identified putative novel regulators... vivo screens in naïve T cells. More broadly, this system can be used to perform in vivo screens in other...power of in vivo screens. Figure 1: Using FITS to perform in vivo CRISPR screens to assess T cell...FITS In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However...However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine...

  37. Plasmids 101: Golden Gate Cloning

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    Blog Post
    ... it easy to create a single gRNA-expressing plasmid, but it can also be adapted to express multiple gRNAs...one 30-minute reaction. The destination vector and entry vector(s) are placed in a single tube containing... appear in the final construct. The destination vector contains sites with complementary overhangs that...TGGA and 3’ overhang TCCG can be ligated into a vector containing those overhangs. Entry DNA overhangs...enzyme and ligase. Although the original destination vector + insert may spontaneously religate, this transient...efficiently clone up to 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages of Golden... step. If your genes of interest or destination vector contain multiple internal restriction sites that...

  38. Plasmids 101: Screening Strategies Used in Plasmid Cloning

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    Blog Post
    ...bacteria that have taken up this vector, but it won’t tell you whether the vector contains the gRNA insert or...on a plasmid, creates a functional 𝛽-galactosidase. Blue-white screens rely on vectors where the gene...deleted, it creates a non-functional 𝛽-galactosidase. Complementing this mutation by expressing this deleted...X-gal allows you to identify cells that took up the vector and distinguish between plasmids that contain the..., you will see two bands: one representing your vector backbone and the other representing the insert....doesn’t contain the insert, you will just see the vector backbone band. Or, you can choose an enzyme that...specific to your insert or by using primers that are vector specific but amplify a potential insert. If you...

  39. Addgene's Top 10 Blog Posts of 2015

    Type
    Blog Post
    ...in CRISPR genome editing.   2. Plasmids 101: Cre-lox Cre recombinase can be used to generate inversions...you can use to express your protein of interest. 4. Plasmids 101: Blue-White Screening Getting a lot of...researchers with many different ways to control gene expression. Get the full scoop from Addgenie A. Max Juchheim... of empty vector contaminants in your cloning experiments and tired of having to PCR many colonies to ...CRISPR/Cas9 and AAV Adeno-associated viral (AAV) vectors are useful for in vivo gene delivery, but have ...Browse Our CRISPR Resources Learn More about Viral Vectors Browse the Repository  ...community members. In addition we’ve experienced incredible growth with our readership expanding from ~10,000...

  40. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ...of Bacterial Expression Systems. Check out our companion post describing protein expression strains, where...Mutation in a chaperonin gene Increases the stability of certain expressed proteins endA, endA1 Endonuclease...mutations Blue/white screening of clones   lacIq lac repressor overproduced, expression from pLac repressed...lab strains designed for subcloning or protein expression. If you were to include customized strains, the...transformation efficiency deoR constitutive expression of genes for deoxyribose synthesis Allows uptake...   TOP10 derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning)....Streptomycin  HB101 derivative. For propagating plasmids expressing the ccdB gene (important in Gateway cloning)....
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