...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...

...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...

...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...

...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...

... 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...

...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...

...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?...

...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...

...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...

...(AAV) is a single stranded, Parvoviridae DNA virus, packaged in a non-enveloped icosahedral capsid, that...designed so that the single stranded DNA folds back on itself to become dsDNA in the nucleus and is a good option...repeats – the sequences that flank the deliverable DNA) are removed, resulting in recombinant AAVs (rAAVs...the type of capsid (outer protein shell) used to package an rAAV vector; these capsids allow the virus to...design single rAAV vectors with multiple genes packaged in them and use elements such as IRES or 2A to...cell machinery, and synthesize a second strand of DNA before expression of their cargo can begin, which...not divide and replicate in the same way the host DNA does. Because of this, fast dividing tissues will...

...analyses needed for packaging AAV. AAV production Overview: The first step in packaging AAV is transfecting...had never worked with AAV, so naturally I had to package some virus for my first experiment. It was a bit... with these tools, I found myself writing AAV production tricks into the margins of my protocol. While...life easier!  In this post, I’ll share some AAV production, purification, and titration tips, while also...transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids to produce AAV...intermediate into the single-stranded genome that's then packaged into AAV’s protein shell, or capsid; 2) the pHelper...plasmid which encodes a transgene of interest that’s packaged into the virus. Two to five days after transfection...

...diligently throughout the production process.  Helper-free production Unlike lentivirus, AAV does not possess...For more details on the production process, check out the Addgene AAV Production protocol. Titering Once...particles via a novel capsid ELISA: Packaging of genomes can limit production of recombinant AAV-2. Gene Therapy...to be digested with DNase before undergoing titering, to remove residual plasmid DNA carried over from ...experience. Here, we’ll go over the basics of rAAV production.  Of course, you will want to make sure that...facilitate efficient replication. The most common production system, however, is a “helper-free” system, in...adenoviral helper genes are supplied on two different packaging plasmids: the pHelper plasmid containing Ad E2A...

... protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection of... into a producer cell line, such as HEK-293T, production of viral particles, and transduction of a target....   Figure 1: Separation of retroviral packaging components onto distinct plasmids for safety.  ...they physically deposit their cargo into the host’s DNA. These viruses also have a propensity to integrate...centrifuging viruses (a necessary step during production), there is a significant risk for aerosol contamination...viruses grown in the lab are gamma retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV)...humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma retrovirus require...

...again! Addgenie Marcy (now our Sr. Director of Production) pie-fully celebrating Addgene's 10 year anniversary...ready-to-use viral vectors from some of our most popular lentiviral and AAV vectors, as well as developing and sharing...expanding our viral vectors service to include AAV Packaged on Request! “I love Addgene. I think resources... recombinant antibodies, another plasmid-based product that, with Addgene’s support, could fill a community... plasmid request came on September 8, 2004, for pcDNA-p160MBP, while the first request for a kit came ...

...library, the authors packaged it into lentivirus and used the resulting lentiviral library to generate ...of recombinant DNA technology has allowed molecular biologists to fuse fragments of DNA together in a ...Saccharomyces cerevisiae, as a key host for the production of their products, including fermented beer and wine, ...simultaneously incorporate multiple single-strand DNA (ssDNA) oligonucleotides (oligos), and thereby rapidly... of dCas9-VPR limits one’s ability to package it into lentivirus and later create stable cell lines expressing...sequence. Should one of these random stretches of DNA contain an enhancer, it will activate its own transcription...Science. 2013. PubMed PMID: 23328393 Wu lab human lentiviral CRISPR library     CRISPR pooled libraries enable...

... host genome. AAVs are limited by their small packaging capacity. Adenovirus Plasmids | Guide Adenoviruses...this genetic information to cells. Adapting plasmid DNA for use with virus-mediated delivery has many advantages...virus infects a host cell, its genome directs the production of more viruses. However, in theory, a viral ...with some having an RNA genome while others have a DNA genome. In addition, some viruses function by permanently...virus type, linked above. Virus Expression Genome Packaging Capacity Virus Size (nm) Cells Infected Target... AAV Transient or Stable* single stranded linear DNA ∼4.5 Kb 18-26 Dividing/Non-dividing No* Very Low ...Low Adenovirus Transient double stranded linear DNA >8 Kb 105 Dividing/Non-dividing No High γ-Retrovirus...

...#61425 - #61427 are intended for lentivirus production : 61425 : lentiviral vector encoding dCAS9-VP64 with... top GeCKO Screening Library GeCKO are pooled lentiviral CRISPR libraries (both human and mouse) for performing...with 2A Blast resistance marker 61426 : lentiviral vector encoding the MS2-P65-HSF1 activator helper complex...complex with a 2A Hygro resistance marker 61427 : lentiviral sgRNA cloning backbone with MS2 loops at tetraloop...SaCas9) that has been human codon-optimized, and packages both the SaCas9 gene and its single guide RNA ...2013 Jan 3. DOI: 10.1126/science.1231143. PubMed . DNA targeting specificity of RNA-guided Cas9 nucleases...

...purify specific regions of DNA, image DNA in live cells, and precisely edit DNA and RNA. The ease of generating... two formats: as liquid DNA, or in select cases, as pre-made lentivirus . DNA libraries are shipped at...homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence...will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue...strands of the target DNA. This results in a double-strand break (DSB) within the target DNA, located ∼3–4 nucleotides...of target DNA. Cas9n increases target specificity. Since two nickases targeting opposite DNA strands are...groove and the non-target DNA strand SpCas9-HF1 - disrupt Cas9’s interactions with DNA phosphate backbone HypaCas9...

...generation packaging system that includes TAT Can be packaged by both 2nd and 3rd generation packaging systems... most lentiviral vectors. Packaging Gag in trans Precursor structural protein of the lentiviral particle...Viral Production FAQ References Glossary Lentiviral Components To increase the safety of lentivirus, the...isoforms of these packaging components. Therefore, lentivirus may not be efficiently packaged by retroviral... Guides Lentiviral Guide Lentiviral Guide...SIN) after integration. Packaging plasmid(s) Envelope plasmid Addgene’s packaging and envelope plasmids ...comprising the 2nd-generation lentiviral system. This system contains a single packaging plasmid encoding the ...

...retrovirus. Packaging using 293T cells This method is very similar to lentiviral packaging methods. The...direct the production of virus with various tropisms. Packaging using helper-free packaging cell lines...Note : While both lentiviruses and γ-retroviruses use the same gene products for packaging (i.e., Gag, Pol...isoforms of these packaging components. Therefore, lentiviruses may not be efficiently packaged by γ-retroviral...γ-Retrovirus Guide Packaging Systems Frequently Asked Questions (FAQ) Glossary γ-Retroviral Packaging Systems The...viral LTRs, differ. As a result, lentiviral and γ-retroviral packaging plasmids are not interchangeable...the viral packaging cell line. For example, Phoenix, a second generation γ-retrovirus packaging cell line...