...Endogenous biotin ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9,...When these three components are co-expressed, the gRNA targets the dCas9 to the loci of interest and dCas9...lines will have varying levels of dCas9 and gRNA expression. Additionally, CAPTURE 3C-based sequencing ...requires the creation of a stable cell line that co-expresses three components: dCas9 with an added biotin ...that is recognized by biotin ligases normally expressed in eukaryotic cells. Using this tag reduces the...CAPTURE 2.0 to be used with primary cells. Find plasmids for CAPTURE 2.0! When compared to the original...conformation data is compared with changes in gene expression, chromatin accessibility, and epigenetic modifications...

...genome editing, activating gene expression, repressing gene expression, and much more. But there’s one...Check out these these plasmids Catch Up on Your CRISPR Background with Our Guide Pages Other Links Check... Lieber A (2018) HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity...CRISPR   Resources on Addgene.org Find CRISPR Plasmids for Your Research Interested in light-inducible...recognition site (Harrington et al., 2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes...interferes and prevents the correct assembly of the crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage...CRISPR activity in many ways such as inhibiting crRNA loading, inhibiting DNA binding, or inhibiting DNA...

...dystrophy. While mini-dystrophin expression levels were lower than expression of wild-type dystrophin in normal...Drawbacks: Transgene expression varies with this approach, ranging from ~10% expression in primary human ... an AAV transfer plasmid Resources on Addgene.org: Check out the Addgene AAV guide Find ready-to-use...this AAV successfully mediated transduction and expression of the full-length transgene in vitro and in ...genomes complement each other to restore the full-expression cassette. AAV’s natural ability to reassemble...overlapping split vector has equal or higher transgene expression when compared to using a single over-sized vector...could have a big impact on the rate of transgene expression with an overlapping AAV since both of these variables...

...out Our CRISPR Guide Pages Find CRISPR Plasmids for Your Research Find Validated gRNAs for Your Research...for future preclinical and clinical work. Find Plasmids for Disease Research on Our Special Collections...myoblasts; when injected into DMD mice, these cells expressed functional dystrophin. Editing genes in vivo is..., the CRISPR components would only need to be expressed for a short period of time, which would prevent...et al. show that the percentage of dystrophin-expressing muscle cells increases over time, and all three...three groups confirm dystrophin expression via Western blot, at <10% of the wild-type level. Muscle histology... reduce off-target editing. Short-term CRISPR expression would be especially desirable, as it would reduce...

...Unpublished or pre-publication plasmids": Unpublished plasmids or plasmids that will not be published are...and entering a plasmid name,plasmid type, and plasmid description/experimental purpose (right). On the...with your plasmids, and adding all the plasmids at once increases the likelihood that the plasmids are given...easy for you to share plasmids. To achieve this goal, we will archive any plasmids you've deposited with...post as a step-by-step guide to the online deposit process. To deposit plasmids online, you will need ...Spreadsheet". Start your plasmid deposit There are two options to begin your plasmid deposit. Figure 2: Choose... Choose to submit plasmid(s) online or by using a spreadsheet. The plasmid information can be entered ...

...in analysis Plasmid-based proteins: plasmid sequence not accurate Check plasmid sequence for frameshifts...important for highly expressed proteins, including “housekeeping” or ubiquitously expressed proteins used for...protein expression is preferable, and more accurate, than normalizing to ubiquitously expressed proteins...lightly marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and... for normalization. Many times the expression levels of these proteins are out of the measurable range...dilutions are in the dynamic linear range (i.e., expression decreases as protein concentration decreases)... expert Review protocol videos for a visual guide to good technique High background Antibody...

...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...after transfecting with the transfer and packaging plasmids. A benefit of the multiple harvest strategy is...on a variety of factors such as the transgene expressed, the viral storage buffers used, and other experimental...will be able to overcome differences in receptor expression. It's all in the mix Even before the lentiviral...infection significantly alters microarray gene expression profiles.” Biotechniques 35.4 (2003). 812-4. ...

... (A) Cumulative CRISPR plasmids shipped by year. (B) Cumulative CRISPR plasmids deposited with Addgene...,000 CRISPR plasmids in our repository, and we have shared almost 300,000 CRISPR plasmids and 12,000 CRISPR...these CRISPR plasmids (and libraries)? We wanted to know, so we classified CRISPR plasmids containing a...CRISPR plasmids requested each year by year of deposit (Figure 3), we saw that once CRISPR plasmids become... CRISPR plasmids seem to never truly go out of style. Instead, an almost even mix of plasmids (by age)...Total CRISPR plasmids deposited by country; square size is proportional to number of plasmids deposited ...Total CRISPR plasmids requested by country; square size is proportional to the number of plasmids requested...

...production. Whether redesigning viral packaging plasmids, investigating different viral production strategies... of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...resources of the Addgene blog Read our beginner's guide to viral vectors Find all of our viral vector blog...Rather than being released into the cell culture supernatant, the viral particles associate with the cell ...production phase, improves adenovirus production." Journal of biotechnology 157.1 (2012): 228-236. PubMed ... trafficking of adeno-associated virus/phage." Journal of Biological Chemistry (2012): jbc-M112. PubMed...

...level and timing of expression can also be affected by the tag.  Protein expression, measured using antibody...Addgene.org Visit the Fluorescent Protein Guide Find antibody plasmids for your experiments ...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...Have you ever wanted to measure expression of your protein of interest in a single cell? Or perhaps, ...applications such as measuring target protein expression levels, assessing post-translational modifications...system (Figure 1).   Figure 1: Cells expressing a particular surface receptor are stained with...interrogated with a fluorescent light source, a cell expressing the associated fluorophore or stained with the...

... in a plasmid with loxP sites such that their expression is turned on or off in cells expressing Cre. ...Addgene.org Check out Our Fluorescent Protein Guide Pages Find Plasmids for Subcellular Localization Find Fluorescent...allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes...efforts have been made to create transgenic mice expressing fluorescent protein reporter constructs in the...above depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after...Introduced to immunology by three papers in the journal Science in 2002. Now, this technique and the in...

... Hub AAV Guide AAV Packaged On Request Browse all in-stock AAV preps Browse all AAV plasmids ...viral capsid, but specificity doesn’t end there: expression from rAAVs also depends on your choice of promoter..., et al., 2006). AAV2 capsids are available as plasmids in the repository and can be selected as a serotype...efficiency in multiple tissues, rapid transgene expression, and can cross blood vessel barriers. It is especially..., et al., 2006). AAV8 capsids are available as plasmids in the repository, as part of our in-stock viral...retinal cells (Issa et al., 2023). AAV9 packaging plasmids are available at Addgene. AAV9 vectors are not...the axon to the cell body and driving transgene expression (Tervo et al., 2016). This functionality makes...

...Addgene.org Fluorescent Protein Plasmids and Resources Fluorescent Protein Guide: Biosensors Keima, another...monitor autophagy, the Pho8Δ60 mutant protein is expressed. Pho8Δ60 localizes to the cytosol in an inactive...yeast Rosella is targeted to the cytosol when expressed without a signal sequence and to the mitochondria...measuring the amount of radioactivity in the culture supernatant (this is indicative of protein degradation). ...

...Additional resources on Addgene.org CRISPR Guide CRISPR Plasmids and Resources CRISPR History and Development...generation of multiple edits or implementation of expression systems to accurately mimic a disease phenotype...best served by this type of targeted design. Alternatively, a disease model can be generated, and drugs...

...Resources ATCC Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen...Invitrogen Cell Culture Basics Guide Abcam Cell Culture Guidelines Resources on the Addgene Blog Learn ...along with Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal...Protein Localization Resources on Addgene.org Find Plasmids for Mammalian Cells ...identification of glioma-initiating cells. The International Cell Line Authentication Committee includes ...cell culture outlined in this article. Now many journals strictly require evidence of cell line provenance...

...the single guide RNA (protospacer+scaffold) in a pol-III (OsU3) promoter driven expression system. We ...all CRISPR plasmids and resources at Addgene Find CRISPR base editors Find CRISPR plasmids for plants ...We use a single polycistronic tRNA-gRNA (PTG) gene system to clone multiple protospacers which enables ...protein, they created a mutant library of E. coli tRNA adenosine deaminases (ecTadA), fused it with dCas9...multiplex editing capability with the endogenous tRNA-processing system." Proceedings of the National ...rice by using Cas9 variants." Plant biotechnology journal (2018). PubMed PMID: 30051586. 9. Hua, Kai, et ...

...observe expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the...transformation Read our Plasmids 101 blog posts Resources on Addgene.org Browse our Plant Plasmids and Resources...page Find synthetic biology plant plasmids Find CRISPR plant plasmids ...way to overexpress a gene and evaluate its function. In nature Agrobacterium contains a plasmid with a... transgenic cultures or plants, our transient expression method enables us to quickly screen and investigate...reporter gene so that the transformation and gene expression can be tracked. During a transient leaf transformation...localization of your protein of interest. Find all plasmids from the Lee-Parsons lab. Test different leaf...

...and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate Cas9-expressing cell lines...Cas9-expressing cell line and then transduce this line with lentiviruses expressing various gRNAs. It’...single-vector system). Tips for using this plasmid Cas9 expression is typically cytotoxic, which makes generation...and transgene expression across different cell types (Aschauer et al., 2013). Expression of hM4D(Gi)-mCherry... injected, the DREADD will only be expressed in cells that express cre recombinase. Typically, this feature...to attain cell-specific expression in transgenic animals, where cre expression is restricted to a particular...delivers gRNA, such as lentiGuide-Puro. The alternative single-vector systems typically express Cas9 and...

...used for. Addgene’s Antibody Plasmid Collection includes plasmids that express antibodies, nanobodies, and...Western blot showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin,...such as parasites or microbes, or to proteins expressed on the surface of cells that have been infected...the protein out of solution, or isolate cells expressing this protein. Read on to learn more about antibodies...context of the immune system, each isotype is expressed at different times of the immune response and ...immunoglobulin genes, recombinant antibodies are plasmid-based. Their sequences can be optimized for specificity...Additionally, the choice of vector can impact the expression level of an antibody (Ayyar et al., 2017). Single-chain...

...substantial: Plasmid tRNA Synthetase Expression Host pMAH-POLY Polyspecific aminoacyl-tRNA Synthetase ...Resources on Addgene.org Find Plasmids for FRET Browse Empty Plasmids to Fluorescent Protein Fusions ...However, since tRNA suppression competes with natural chain termination at stop codons, expression levels of...Xenopus oocytes, mainly). Nevertheless, tRNA suppression plasmids are available from depositing labs that...Fig. 1) which can be carried out using Addgene plasmids. His-tag labeling reagents You may already have...Fusions Check out Our Fluorescent Protein Guide ...Davis, 2015). By using a novel aminoacyl tRNA synthetase/tRNA pair, a researcher can lure the ribosome...