... are, in most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet ...infection; however this would not be great for a gene therapy vector! For viral vectors, only the protein... is (thankfully) gutted of many essential viral genes. How it is, exactly, that heaps of viral vector ... particles are made while missing the polymerase gene may seem like a mystery. But it’s simple. The RT...the only component that is not dependent on viral genes. It is derived from the host/producer cell membrane...full “life cycle” of expression of selfish, lazy genes of a non-living viral particle, and how we as dedicated...Kantor, B. (2021). Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State...

...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https...conditions commonly encountered in human gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https...Grames, M. S., & Klein, R. L. (2018). More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose–...dose–response and comparison to AAV PHP.B. Gene Therapy, 25(5), 392–400. https://doi.org/10.1038/s41434-018...Adeno-Associated Virus Serotype 9 (AAV9) Preparations. Human Gene Therapy Methods, 30(6), 214–225. https://doi.org...Resources on addgene.org  Download Addgene's Viral Vectors 101 e-book Additional Resources on the Addgene Blog... by Addgene in 2021 asking scientists what data would improve our AAV Data Hub.   The Addgene Data...

...functionally characterize combinatorial gene knockouts generated via multiplexed gRNA expression (Wong ...et al., 2014; Konermann et al., 2014) individual genes for studies interrogating their functions. The continual...list of effective gRNA sequences targeting your genes of interest. Thanks to the tremendous efforts made... et al. "Genome-scale CRISPR-mediated control of gene repression and activation." Cell 159.3 (2014): 647... "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities." Cell ...Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death...incredibly useful for the efficient generation of individual (and multiple) genetic perturbations in single cells...

...dominant disease-causing gene, or that carry only one copy of a recessive gene. Ma et al. claim that CRISPR...Variants Target Multiple Genes at Once with CRISPR Resources on Addgene.org Browse All CRISPR Plasmids...authors to title the paper “Correction of a pathogenic gene mutation..” rather than using term ‘editing.’ While...but this advantage likely won’t transfer to all genes we’d like to target. In addition, as on-target editing...Reference Ma, Hong, et al. "Correction of a pathogenic gene mutation in human embryos." Nature. Epub 02 August...hypertrophic cardiomyopathy and other genetic disorders, pre-implantation genetic diagnosis (PGD) screens embryos...PubMed PMID: 28783728. Additional Resources on the Addgene Blog Check out Our New CRISPR Topic Page Read ...

...detect gene copy number based on the number of fluorescent puncta observed. By labeling two genes of a ...vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed targeting...Chen et al. successfully labeled protein-coding genes with both intronic and exonic gRNAs. In fact, the...also monitor the spatial relationship of the two genes over time, at a range of distances from 2-75 Mbp... envision using CRISPRainbow in combination with gene editing. CRISPRainbow requires very short, 11-mer...plasmids from this publication at Addgene. Additional Resources on the Addgene Blog Read More about Fluorescent...cells! This technique was developed in the lab of Addgene depositor Stanley Qi and used in Chen et al. to...

...label cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate...used LexA and rTetR systems such that they activate gene expression from different promoters. Moving to an.... GFP expression robustly activated the reporter gene TdTomato, whose expression was absent without electroporated...sites. Placing a lox-STOP-lox cassette upstream of a gene of interest blocks transcription when Cre is absent...present, it removes the STOP cassette, activating gene expression. Although Cre isn’t a modular protein...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed...commonly used in optogenetics, opening up the possibility of adapting GFP lines for optogenetics experiments...

...deletion of one or several of these genes permits the expression of genes farther downstream in the construct... two tandem sets of genes aligned head-to-head and the deletion of one of the gene pairs. Brainbow 2.0...Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized ...organized in tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated... which uses Cre to trigger the inversion of two genes aligned head-to-head. Another variation, Brainbow...located directly upstream of the fluorescent protein genes, Cre recombination is required before fluorescence...fluorescent proteins topic page Resources on Addgene.org Browse Addgene’s fluorescent protein collection Learn...

...human genes have counterparts in the rat (Gibbs, et al., 2004). Additionally, breakthroughs in gene modification...abundance of genes related to limb regeneration (Vieira et al., 2019). To study regeneration scientists ...during and after embryogenesis. Addgene has over 600 plasmids that contain Xenopus genes and a tool-kit ...models.  Check out Addgene’s collection of plasmids containing Rattus norvegicus genes! Mexican axolotl ...array of molecular tools, such as gene manipulation and mutagenesis techniques, are available to the scientific...world worked to characterize each gene’s function. Some key genetics research with Arabidopsis include... for scientists to study human disease-relevant genes in such a controlled environment displays how essential...

...you were cloning a gene into an expression vector, you would want the start of the gene to end up just downstream... 5’ of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you can then ...leaving the 5’ end of your gene just downstream of the promoter and placing the gene in the proper orientation... multiple sites allows you to easily orient your gene insert with respect to the promoter. For example...restriction sites EcoRI, XhoI, and HindIII. To place your gene in the proper orientation downstream of the promoter...binds the plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily...green, insert) are cut with restriction enzymes to generate compatible overhangs that allow them to bind. ...

...the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection of cells...to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone...had a point mutation in the puromycin resistance gene that made it less effective in certain cell lines...Resources on the Addgene Blog Browse Our CRISPR Posts Learn about Using CRISPR to Generate Knockouts Read...Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified... the key component of most CRISPR systems and generates double strange breaks at target sites 5' of the...your 20 nucleotide guide sequence of choice (for generation of a gRNA that will target SpCas9 to the approrpriate...