...Joachim Goedhart’s article on the Addgene blog: Fluorescent Proteins 101: GFP Fusion Proteins – Making the ...protein of interest. The green fluorescent protein (GFP) and mCherry, two of the most common tags currently...References on Addgene.org Check out Addgene's Chemogenetics Guide Find fluorescent proteins at Addgene ...on the Addgene blog Browse Fluorescent 101 blog posts Read about chemogenetics vs. optogenetics Find more...Emory University School of Medicine. Optogenetics and chemogenetics are powerful tools to modulate the ...neurons and other brain cells. Since the opsins or chemogenetic receptors used in these techniques cannot be...versatile method to identify the expression of transgenes of interest. Furthermore, fluorophore variants...

...cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic...alternative method for generating transgenic mice strains and for non-invasive in vivo gene editing. The bottom...repression, transgenic mouse line creation, and in vivo gene editing of mice, with the potential to be used for... Primary cells are often refractory to gene delivery methods, but the researchers was able to edit hiPSCs...transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~50% Human HSCs  Myd88  ~50% Table 1: Nanoblades...cells when using a gRNA targeting the Fto gene, while editing was not detected in cells electroporated with... used Nanoblades to disrupt the tyrosinase (Tyr) gene in mouse zygotes, which results in albinism in mice...

...plasmids described in this work including the soCoChR-GFP plasmid as well as several derivatives encoding alternative...ShadowG, a novel dark green fluorescent protein (GFP) with an absorption peak of 486 nm and ShadowY, a... to the Hot Plasmids episode! Expanding the optogenetics toolbox with CRY2clust Article contributed by...Basic Science (IBS) has developed another useful optogenetic tool that enables robust and efficient oligomerization...CRY2clust, when integrated into a Ca2+ -modulating optogenetic system (OptoSTIM1), caused two-fold faster changes...soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego  Listen ...Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool used by neuroscientists to...

... FRET tools, double dimerization GFPs, and circular permutation GFPs. Hopefully we will have new tools...her work has always been focused on gene transfer, from transgenes to plants, chloroplast to the nucleus...easy to express genes in, which makes it a good laboratory workhorse. Tips & tricks Addgene: Do you have ...The MoClo Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose... Now there are many, so if you want to make a 10-gene construct, you don’t have to worry about repeating...constructs to allow people to, for example, knock out a gene with simultaneous complementation, with a knockout...Marillonnet share with the scientific community via Addgene, the struggles of plant scientists and how they...

...where his group focuses on gene regulation in nematode molting and spermatogenesis. His group also builds ... one of the model organisms which I use to study gene regulation during developmental processes, CRISPR...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...Craig Mello’s lab – used inactivation of the unc-22 gene as their selection marker (Kim et al., 2014). Meanwhile...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this... I was able to knock-in a 2xFLAG epitope into my gene of interest, I encountered low efficiency (0.13%...efficiencies, obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence...

...library - Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by... hSyn FLEx mGFP-2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance...  After validating this method through monitoring GFP degradation, this protein compartment was used to...a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a compact Cas9 ortholog from Staphylococcus...targeted synaptophysin tagged eGFP. AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE from Hongkui Zeng's lab... Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr et al Cell., 2020....scFV library and characterized them by ELISA, homogeneous time resolved fluorescence, and suspension bead...

...with a fluorescent reporter, such as GFP or mCherry, through genetic modification. As your protein of interest...overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore... you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in...Resources on Addgene.org Addgene's Antibody Guide Ready-to-use antibodies in the Addgene repository Antibody...vector also encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45...-Cy7. Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need...cytometer which channel to use to detect eGFP. As you saw in Figure 4, eGFP strongly overlaps with FITC, hence...

...red light instead of blue to excite a molecule of GFP. Because red light has approximately half the energy...light, two photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point...infinitely small point of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot...PubMed PMID: 26186186.  Additional Resouces on the Addgene Blog Lightsheet Microscopy Choosing Your Fluorescent...Fluorescent Protein for Your Experiments Resources on Addgene.org Browse Our Fluorescent Protein Resources Find...Fluorescent Protein Backbones Find Plasmids for Optogenetics ...

...overexpressing a gene of interest in the plant. A common method for silencing genes Virus induced gene silencing...by replacing the genes on the T-DNA region with our gene of interest or a reporter gene so that the transformation...transformation. Choose good reporter genes There are many great reporter genes available and which one you choose...transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase) with histochemical...Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants." Plant...Hikmet Budak. "Virus-induced gene silencing, a post transcriptional gene silencing method." International...stable cell line or plant is regenerated from the transformed tissue. Gene function is commonly studied...

...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...neurons and GFP (green) to label neurons transduced by each serotype. Working at Addgene has shown ... cassette with an ubiquitous promoter to express GFP in a group of young rats. We found that, in this ...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...

...stem cells By Jennifer Tsang CRISPR gene interference (CRISPRi) and gene activation (CRISPRa) are great ways...GABA-ergic interneurons. These include mDlx-GFP, hDlx-FLEX-GFP, hDlx-Flex-dTomato, mDlx-GCaMP6f, hDlx-GiDREADD-dTomato...the plasmids at Addgene! A new tool for synthetic biology uses CRISPRi to control gene expression. This...nucleus and modulate transcription of the mCitrine gene. mCherry is constitutively expressed. Cells are ...selectivity and found that the best motif was from the Lck gene. For marking microtubules of mammalian cells, the... ways to modulate the expression of nearly any gene via specific guide RNA (gRNA) sequences. But these...-IgG1-Fc, Addgene #140700) pMAK463 (NBαMouse-IgK, Addgene #140701) pMAK464 (NBαEGFP, Addgene #140702) ...

...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...from transient overexpression of protein fusions to gene-edited cells can decrease the number of fluorophores...Fluorescent Protein Should I Use? Resources on Addgene.org Generate Fluorescent Protein Fusions Request Plasmids...in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion with your... dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine (Cys4) peptide tag could...TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that reacts specifically...

...protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes... I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein Guide Expression Vectors from... overexpress a recombinant protein? It is not generally in a cell’s best interest to overexpress a protein...for affinity and purification An affinity tag, generally a relatively small sequence of amino acids, is...interest would eventually get ridiculous (you generally don’t want more tag than protein), 2-3 tags is...using plasmids found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly...purification is provided by GE Healthcare Life Sciences. Addgene pages and blog posts of interest: Which Fluorescent...

...mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories later...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed... based on using a thin plane (sheet) of light generated by sunlight to observe single gold particles with... nerve afferents labelled with a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical...).  These viral plasmids are available through Addgene.  The combination of LSFM with multiple tissue ...

...antibodies against popular fluorescent proteins (GFP and YFP) and protein tags (c-MYC and HA). We have...ways of generating antibodies, for example through the use of less toxic adjuvants; 2) generating complex...and 4) generating mAbs against new cancer biomarkers. We believe that repositories, such as Addgene and ...The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years Data Freedom: The Expansion of...

...Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used to study gene expression...for visualizing gene expression in vivo.  But what ultrasound lacked was reporter genes that could produce...Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when expressed, enable cells...vesicles gene clusters from a diverse set of gas vesicle-expressing species. The gas vesicle genes from Serratia...structural gene gvpA marked with EBFP2 and the second cassette contains the P2A-linked accessory genes gvpNJKFGWV...first-generation mammalian ARGs (mARGs) enabled ultrasound visualization of in situ mammalian gene expression...hypothetical protein, improving expression, and named this gene set bARGSer, for bacterial ARG from Serratia sp....

...REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large gene into smaller...the introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated in an AAV vector (Figure...Figure 5: A) Representative confocal images showing GFP expression from ssAAV5s with hIBA1 or hIBA1a promoters...Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s. C) Quantifications...doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav...AAVs, and then splicing their mRNAs, the functional gene product is reconstituted within the target cells...

...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... lines. Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9 and further...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...

... time or location can be critical when, say, the gene is essential during development and a knockout is...activity can be monitored by phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing...membrane-targeted SuperPLDx30 (magenta) and PA probe GFP-PASS (green). After 30 minutes of blue light illumination...the Addgene blog Viral Vectors 101: Optogenetics Additional resources on Addgene.org Optogenetics Guide... The Chow Lab has also generated a variety of membrane-recruitment optogenetic tools for manipulating ...a full toolbox and workflow for generating new Rho GTPase optogenetic systems (Berlew et al., 2022). Stopping...LOV, CRY2-CIB, and other optogenetic modules? Or want to find more optogenetic tools in our collection?...

...translational. Ex: the discovery of GFP. Chapter 4: Goals of the Center for Regenerative Meidince - Understanding...Additional Resources from Addgene Learn about Retinal Gene Therapy from Connie Cepko Find Plasmids from the...treat disease through tissue repair, stem cells, gene therapy, and tissue engineering. How the multidisciplinary...Production Specialist at Addgene. He is the audio engineer and producer for the Addgene Podcast.     Additional...Darrell Kotton is the director of the Center for Regenerative Medicine at Boston University Medical School...source biology in his own lab, at the Center for Regenerative Medicine, and within the biomedical research...Along these lines, Darrell recently joined the Addgene Board of Directors where he hopes to help keep ...