... Dual lentivirus system with ubiquitous expression of sgRNA (top) and neuron-specific expression of dCas9... for generating the SPH mouse. (B) AAV plasmids used to express CRE under neuron-specific promoters (aCaMKII... (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH activator paired...neuronal populations.  The Yang Lab’s AAV plasmids used to express Cre under neuron-specific promoters, including...Savell et al., 2019). This dual lentivirus system pairs ubiquitous expression of sgRNA(s) and mCherry (vector...without inducing expression of the non-targeted variant. Moreover, isoform-specific expression of Bdnf resulted...comprehensive CRISPR guide Find CRISPR plasmids and resources Find CRISPRa plasmids ...

... Protocols Lentivirus Production Lentivirus Production You may also ... 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ... each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...endotoxin removal step. For high quality plasmid DNA, the plasmids should also be propagated in an endonuclease... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be... 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios...this protocol to generate lentivirus Protocols...

...could lead to reduced transgene expression over time, as the lower expressing clones take over the polyclonal... pool of stable cells. Transducing cells with lentivirus results in a heterogenous polyclonal population...that are more likely to retain stable transgene expression. Other methods of generating a monoclonal cell...protocol for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation...protocol for generating stable cell lines with lentivirus . Pro-Tip Because the polyclonal cell pool is...population be monitored regularly for transgene expression. This protocol has been optimized for adherent...sufficiently expanded, screen the lines for transgene expression and/or other phenotypes. For example, perform...

...Pooled Libraries All Viral Plasmids Packaging Plasmids Transfer Plasmids Lentiviruses, a subset of retroviruses... Viral Plasmids Lentiviral Plasmids Lentiviral Plasmids Additional Resources...among multiple plasmids. Some plasmids (typically called packaging and envelope plasmids) encode components...packaging plasmids that can be used with 2nd and 3rd generation lentivirus technologies. ID Plasmid Generation...generation transfer plasmids Trono 12259 pMD2.G N/A Envelope VSV-G-expressing envelope plasmid Trono 8454 pCMV-VSV-G...envelope plasmid Nienhuis and Salmon 35616 pCAG-VSVG N/A Envelope VSV-G-expressing envelope plasmid Nienhuis...lentiviral plasmids to express your CRISPR gRNA? Please see our CRISPR Empty gRNA plasmids table and search...

...restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just ...correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly correct. Because...Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based) Mammalian Lentivirus pRDA_722...pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid pRDA_791 (contains only orio and...Mammalian or drosophila) Delivery type (lentivirus, AAV, transposon, Plasmid) Destination vector (dropdown options...the assembled plasmid. It shows you each component, with the ID of the component plasmid, component name...the component plasmids on the Addgene website. Step 4: Naming your assembled plasmid and generating...

...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral.... coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ...against unique regions of the transfer plasmid.   Lentivirus resources Browse lentiviral preps available...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes reported...mL, or both. Two days later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was...was not detected in the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre...

...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety...viruses grown in the lab are gamma retrovirus, lentivirus, adenovirus, and adeno-associated virus (AAV)...

... Viral Vector Packaging Service Lentivirus Browse In-Stock Lentivirus Pooled CRISPR Libraries Cas9 Pooled...Barcoding Libraries Control Addgene's lentiviruses are made from plasmids that are currently in our repository... list of gRNA plasmids targeting specific genes. For more information on CRISPR plasmids and function,...library expresses EGFP for easy visualization via direct fluorescence. Morris Control Lentivirus Browse... of available viruses based on plasmids in our repository that express fluorescent proteins. ID Name Insert...Ready-to-use lentivirus available from Addgene's viral vector packaging service. Browse constructs currently...repository. You can obtain the plasmid from us or receive a ready-to-use viral preparation from the inventory...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...need. If you'd ever like to write about a recent plasmid deposit please sign up here.        A retroviral...However, some cell types are poorly transduced by lentivirus, and thus screening with available CRISPR libraries...Primary murine T cells cannot be transduced by lentivirus and have been difficult to manipulate genetically...type 2 (Th2) cells from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation...differentiation and activation, using expression of differentiation factors as the screen readout. This retroviral...

...cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via...pools of A549 cells transduced with Cas9 expressing lentivirus at the indicated multiplicity of infection...wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects growth rate...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but...thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low (even in...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%...

... Plasmid Collections Plasmids for Stem Cell Research Plasmids for Stem Cell Research...Reprogramming Plasmids Differentiation and Transdifferentiation Plasmids Stem Cell Research Plasmids Stem cells...utilize plasmids to increase expression of key factors. The tables below will help you find plasmids available...information page. Reprogramming Plasmids Browse the table below for plasmids used to create induced pluripotent...Method Species Description Article PI Lentivirus Human Expression of human Oct4, Sox2, Klf4, Myc, and Brd3R...Commun. 2018 Jul 6;9(1):2643. Otonkoski Lentivirus Human Expression of human Sox2, Nanog, Oct4, and Lin28...318(5858):1917-20. Thomson Lentivirus Human Doxycycline-inducible expression of human Oct4, Sox2, Klf4,...

... 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...mutagenesis, transgene reactivation, residual expression Lentivirus Integrating High Low Efficient and Stable...reprogramming. Additionally, lingering expression or re-expression of viral transgenes in iPSC-derived cells...cassette that expresses multiple reprogramming factors driven by one promoter. While lentivirus has similar... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...

...the transfer plasmid, a plasmid encoding Rep and serotype-specific Cap genes, and a plasmid encoding adenoviral... coli . In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits. Resources...viral production methods and quality control for lentivirus and AAV. Viral...AAV Production Titer Quality Control Resources Lentivirus Production Titer Quality Control Resources Overview...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes reported...mL, or both. Two days later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...of bacteria that can cause disease in humans. Expression of this system is tunable via a rhamnose-inducible...lentiviral capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced...packaging Cas9/gRNA ribonucleoproteins for transient expression. They replaced the tetraloop of the sgRNA scaffold... aptamer binding protein for packaging into a lentivirus-like particle. Read the paper in Nucleic Acids...Acids Research Find the gRNA and packaging plasmids   Cytosine deaminase for RNA editing Feng Zhang’s ...

...packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap plasmid which provides...AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not...protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes which help mediate AAV...going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs which are...AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple ways to ...multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR...

...from Rudolf Jaenisch’s lab in plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 ... are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation...modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear....that help package DNA, were associated with gene expression or silencing. To alter DNA modifications, researchers...Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and...well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks...cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation...

... the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors. Lentiviruses are sometimes...instead get expression of the sensor, marker, or whatever other tool you chose to express. In the case...that the virus must have its structural proteins expressed from a reverse-transcribed piece of DNA called...is done, and the transgenic genome will simply express from whatever promoters were included in the vector...And there you have it, the full “life cycle” of expression of selfish, lazy genes of a non-living viral ...Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B. R. (...

... to find plasmids that can be used to engineer various types of viruses. Lentivirus Plasmids | Guide Lentiviruses...enabling long term transgene expression. Overview of Viral Plasmids While plasmids are typically used to encode...for select plasmids are now available through Addgene, learn more here . Browse Viral Plasmids by Type Visit...direct high but transient transgene expression. γ-Retrovirus Plasmids | Guide γ-retroviruses, commonly referred...organism. Figure 1. Using viral plasmids to generate virus. A) Viral plasmids have been engineered such that...genomes have been adapted into a plasmid-based technology, such that plasmids can be used to create viruses...researchers can introduce plasmids to a host to generate virus. Furthermore, these plasmids can be modified to...

...vectors and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G.... the envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini...(PX461) expresses both a Cas9 protein and a gRNA. But instead of Cas9, this plasmid expresses Cas9n (a...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and...Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid. Envelope proteins determine which types ...wide range of applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB...BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an...

...permanently induce expression? Check out our Retro expression vector and Lenti expression vector. Turn it...ablate protein expression. If you need the ability to control when you turn off expression for good, the...inducible expression (more on this below) will both achieve temporary repression of protein expression. Addgene... a gene (cDNA) can be done by either plasmid or virus. A plasmid carrying your cDNA can be targeted for...appropriate promoter to express your cDNA, depending on the level of expression you would like. Check out...choosing. Viral expression is another mechanism to integrate your choice cDNA. Both lentivirus and retrovirus.... Whether you want to express or repress, good luck on your protein expression journey! Resources and...