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We narrowed to 43 results for: lentiviral production packaging dna

Showing: 21 - 30 of 43 results

  1. Empty Backbones - Choosing Your Perfect Plasmid Backbone

    Type
    Collection
    ... - Tet-inducible lentiviral Gateway destination vector for gene expression Lentiviral backbones made by...collection of plasmids for packaging and expression, see our dedicated Lentiviral Plasmids page Retroviral... Tet-inducible lentiviral shRNA expression pLKO.1 - TRC cloning vector - Lentiviral shRNA expression...Blasticidin Mammalian pLX304 - 3rd gen lentiviral vector lentiSAMv2 - Lentiviral sgRNA cloning backbone pLKO.1-...Localization pcDNA3-EGFP - C-terminal GFP for mammalian expression pLV-mCherry - 3rd gen lentiviral plasmid...Ubiquitin pYPQ203 (pMDC32-Ubi1) - Multisite Gateway T-DNA entry plasmid (attR1 & attR2); Zea mays Ubi1 promoter...fusion protein. This means that the final product should be translated as a single string of amino...

  2. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...measure the DNA concentration, use 1 μL) 2 μL 10x NEB T4 DNA ligase buffer 1 μL NEB T4 DNA ligase add ...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles...#B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha ...will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick gel extraction kit. Elute...kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB ...transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely ...optimal puromycin concentration F.3 Protocol for lentiviral infection and selection G. Safety H. References...

  3. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect dividing and nondividing...Excisable Cre-lox lentiviral vectors: This approach is the same as using a standard lentiviral vector, but ...nondividing cells and have an ~8kb packaging capacity. With this packaging capacity, reprogramming factors...generated from EBNA1 iPSCs still had episome DNA present. DNA-Free Methods RNA Delivery: For this approach...Need to verify loss of minicircle RNA Delivery DNA-Free High High Transgene-free and vector-free Multiple...Multiple transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free. Slow...or multiple single-gene vectors can be used to package reprogramming factors. An important milestone of...

  4. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    ...inventory to 25 lentiviruses and 25 AAVs. These viruses have been distributed in over 200 packages to more than...work. After all, as we like to sayat Addgene, productivity is infectious. Curious which viruses researchers...lentiCas9-Blast Most of Addgene’s lentiviral inventory is based on CRISPR tools, though we do provide a... Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast. Purpose...requested lentivirus and AAV of 2016. The top viruses of 2016 were (drumroll please)... Top Lentivirus: lentiCas9... Cas9-expressing lentiviruses will no longer be carried over to the gRNA lentiviruses. This means that...lentiCas9-blast lentivirus from Addgene, you could use it in conjunction with gRNA lentiviruses easily made...

  5. Plan Your Experiment

    Type
    Collection
    ...high efficiency Lentiviral transduction Cas9 and gRNA can be present in a single lentiviral transfer vector...positive cells Packaging and envelope plasmids provide the necessary components to make lentiviral particles...amplified, PCR products are denatured-renatured, treated with a nuclease that cleaves DNA heteroduplexes...particles (for details, see our AAV Guide ) ∼4.5 kb packaging limit (only compatible with smaller Cas enzymes... between your gRNA targeting sequence and target DNA may result in reduced cleavage. The number of alleles...increase the likelihood that a nonfunctional protein product is produced. Alternatively, gRNAs can be designed... is absolutely necessary for Cas9 to bind target DNA. As such, one can start by identifying all PAM sequences...

  6. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    ... its packaging capacity is limited to ~4.7kb, or roughly half the packaging limits of lentiviral and adenoviral... up packaging an oversized genome of ~9 kb into AAV. They found that in defiance of the packaging limit...determined that even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb... But despite this, larger functional transgene products were produced following transduction.  So what...oversized AAV genomes are fragmented or truncated when packaged and then following transduction, the partial genomes.... Each piece of the gene is then independently packaged into an AAV. When a cell is co-infected with both...reconstituted after integration; and a sequence of stuffer DNA after the gRNA target site. Donor B contains: 400...

  7. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ...sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from...error, fragments of DNA from the helper plasmids or the cell genome can be packaged inside the vectors ...determine the identity and serotype of the packaged DNA and look for potential contaminants. The analysis is ...mentioned earlier, it is common to find DNA from the packaging cell genome, bacterial genomes, cloning...for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression...used to identify DNA contaminants in AAV preparations and provide information on these DNA species with exquisite...What if bacteria snuck into the prep during the production process? These questions are especially important...

  8. Viral Vectors 101: Transductions

    Type
    Blog Post
    ...viral packaging elements that will ensure the virus gets produced, and viral particles are packaged, when... transfected along with viral packaging plasmids into a viral production cell line, usually HEK-293 cells...expressed within the production cell line, viral particles will be produced and packaged, resulting in functional...with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired DNA to be integrated can be...bacterial in nature. All three of these methods involve DNA incorporation into the cell, but the mechanisms vary...Typically, one day after transfection into the production cell line (depending on the virus), virus can...Services Resources on the Addgene blog Viral Production at Addgene Viral Vectors 101: What is a Virus?...

  9. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    ...synthesize complementary DNA copies of their genome upon infecting a host cell. This viral DNA (also called proviral...-studied lentivirus is HIV, which many lentiviral tools have been derived from. Lentiviruses have slightly...larger packaging capacities than gamma-retrovirus, coming in around 9 kb. Unlike gamma, lentivirus can infect...proviral DNA) is then inserted into the host's genome and is used to make more virus. The most studied ...Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some other ...can replicate and divide along with the endogenous DNA. AAV also has low immunogenicity, meaning it does...Adenovirus Adenovirus has a linear, double stranded DNA genome which is non-enveloped. It can infect humans...

  10. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...Central PMCID: PMC4778041. Find CRISPR and lentiviral packaging plasmids used in this publication at Addgene...this approach could permit some viral protein production, so researchers are also interested in precisely... with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy...Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-cells to...level of protein structure, CRISPR gRNAs require DNA sequence-specific binding. Patients’ HIV-1 genomes...only after stable expression of Cas9/gRNA. Viral production is higher in this model than in latent HIV-1 ...Rice CM, Bhatia SN. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep...
Showing: 21 - 30 of 43 results