...Calcium sensor in astrocytes (GCaMP6f), cytosolic Conditions and constraints for astrocyte calcium signaling... inverse fluorescent biosensor for citrate (Citron/Citroff), mitochondrial or cytosolic High-Performance...proteins. Genetically encoded biosensors can be introduced into cells, tissues, or organisms for long-term... with different binding affinities or negative-control constructs used to characterize the sensor's behavior...differ from the original characterization (e.g. in vitro vs. in vivo , cytoplasmic vs. extracellular vs. ...295-300. Douglas Kim , GENIE Project Calcium Bicistronic vector expressing GCaMP6 and mRuby2 for imaging... GCaMP6f in PV cortical interneurons under the control of the E2 regulatory element Viral manipulation...

...Patrick Aebischer and Didier Trono Constitutive Retroviral shRNA Retroviral vectors for constitutive shRNA... digest) David Root 10879 pLKO.1 ‐ TRC control Negative control vector containing non-hairpin insert David...Lentiviral Tet-regulated Lentiviral Constitutive Retroviral Resources Additional Resources: Human and mouse...David Root 1864 pLKO.1 ‐ scrambled shRNA Negative control vector containing scrambled shRNA David Sabatini...using the 2nd generation packaging system. Didier Trono Cre-regulated Lentiviral shRNA Lentiviral vectors...subcloned from pLVTHM) On Patrick Aebischer and Didier Trono 11779 pLVCT-rtTR-KRAB-2SM2 2nd generation; Transgene...subcloned from pLVTHM) Off Patrick Aebischer and Didier Trono 11644 pLVET-tTR-KRAB 2nd generation; Transgene (...

...Production Titer Quality Control Resources Lentivirus Production Titer Quality Control Resources Overview of...subunit (RPP30) as a control for normalization purposes are being used. Quality Control Mycoplasma The 293T...Addgene's viral production methods and quality control for lentivirus and AAV. Viral...virus preps are titered and subjected to quality control by Addgene before being distributed to customers...production protocols, titering methods, and quality control are described below. AAV Production AAV distributed...probes targeting the ITR elements and an internal control of known titer (protocol modified from Lock et ...performed by droplet digital PCR (ddPCR). Quality Control Addgene ensures high quality viral vectors by optimizing...

...murine) ecotropic retroviral transforming sequence C-CBL, CBL2, RNF55 CBLB Cas-Br-M (murine) ecotropic retroviral...TSHA CGB chorionic gonadotropin, beta polypeptide CGB3, hCGB CGB1 chorionic gonadotropin, beta polypeptide... 1 - CGB2 chorionic gonadotropin, beta polypeptide 2 - CGB5 chorionic gonadotropin, beta polypeptide 5...chorionic gonadotropin, beta polypeptide 7 CG-beta-a, FLJ35403, FLJ43118 CGB8 chorionic gonadotropin, beta... CKLFSF8-V2 CNTF ciliary neurotrophic factor HCNTF CNTFR ciliary neurotrophic factor receptor MGC1774 ...14, CST-17, CST-29 CRH corticotropin releasing hormone CRF CRHR1 corticotropin releasing hormone receptor...CSH2 chorionic somatomammotropin hormone 2 CS-2, CSB, hCS-B CSHL1 chorionic somatomammotropin hormone-like...

...Biology CMV-LUC2CP/intron/ARE, CMV-LUC2CP/ARE : A splicing reporter that contains an intron in the middle ...in assays ranging from use as a reporter gene in vitro and in vivo to use in biosensors. The most common...expression constructs available that are driven by a strong constitutive promoter and can be used to monitor...created by fusing NanoLuc® and Venus fluorophore to Troponin C Ca 2+ binding domain. Nano-lanterns , Enhanced...pKC-4.06 : Nonsense-mediated decay reporter and control plasmid utilizing Firefly luciferase. Transcriptional...luciferase gene fusions. Renilla luciferase under the control of a CMV promoter is present for normalization ...Vector Firefly Vector for investigating regions controlling transcription Debrya Groskreutz 60323 pGL4.23...

...transfer plasmids Trono 12259 pMD2.G N/A Envelope VSV-G-expressing envelope plasmid Trono 8454 pCMV-VSV-G...1a-driven GFP and shRNA under the control of a tet-responsive H1 promoter Trono 11651 pLVUT-tTR-KRAB 3rd Inducible...transgene expression, contains intron that gives you high expression. Trono 12254 pWPI 2nd EF-1alpha driven... Transfer Plasmids Lentiviruses, a subset of retroviruses, are some of the most common and useful types...plasmids along with additional packaging components Trono 63586 psPAX2-D64V 2nd Packaging For making Integrase...transfer plasmids along with a plasmid encoding Rev Trono 12260 psPAX2 2nd Packaging Contains Gag, Pol, Rev...used with 2nd and 3rd generation transfer plasmids Trono 12263 pCMV delta R8.2 2nd Packaging Contains Gag...

...Badran 212125 pBK-Ma pylRS nitroY/haloY-F5 PylRS M. alvus 3-nitrotyrosine (3-nitro-Y), 3-halotyrosine (3-halo-Y...synthetases are introduced into the cell on plasmids and the new amino acid is introduced in the media. ...Mehl 85498 pDule-3-nitroTyrosine (5B) 3NY (5B) synthetase M. jannaschii 3-nitroTyrosine Bacterial TAG Ryan...Mehl 85499 pDule2-3-nitroTyrosine (5B) 3NY (5B) synthetase M. jannaschii 3-nitroTyrosine Bacterial TAG Ryan...Chin 174078 pDule-3-nitroTyrosine (A7) 3NY (A7) synthetase M. jannaschii 3-nitroTyrosine Bacterial TAG Ryan... 174079 pDule2-3-nitroTyrosine (A7) 3NY (A7) synthetase M. jannaschii 3-nitroTyrosine Bacterial TAG Ryan...cognate charged tRNA. Once these plasmids have been introduced in the cells, the non-canonical amino acid can...

...Boyden Voltage Reporter: Voltron 119036 pAAV-hsyn-flex-Voltron-ST Syn Voltron-ST none Cre dependent 1 ...filtering options. *AAVrg = retrograde serotype, produced with the AAV retro helper plasmid from the Karpova...proteins, including calcium sensors, under the control of various promoters. Sensor Green Calcium Sensors...Oxytocin (OT) Sensors GRAB_OT Voltage Sensors Archon Voltron JEDI-2P ATP Sensors iATPSnFR2 5-HT Sensors GRAB... dependent) Serotype AAV1 AAV2 AAV5 AAV8 AAV9 Retrograde AAV PHPeB Clear Filters ID Name Promoter Sensor...pAAV-hSynapsin1-axon-GCaMP6s-P2A-mRuby3 Syn axon-GCaMP6s bicistronic mRuby3 Constitutive 5, 9 Tian 112010 pAAV-hSynapsin1... 140555 pAAV-hsyn-GRAB_DA-mut Syn GRAB_DA-mut (control) none Constitutive 9 Li 140556 pAAV-hsyn-GRAB_rDA1m...

...CRISPR Libraries Cas9 Pooled Barcoding Libraries Control Addgene's lentiviruses are made from plasmids that...76,441 unique sgRNAs along with 1000 non-targeting controls. This backbone does not contain SpCas9. 76,441...,114 human genes along with 1000 non-targeting controls Puromycin Doench , Root 73179 Human gRNA pooled...76,441 unique sgRNAs along with 1000 non-targeting controls. This backbone contains SpCas9. SpCas9 and 76,...,114 human genes along with 1000 non-targeting controls Puromycin Doench , Root Human activation pooled...57,050 unique sgRNAs along with 500 non-targeting controls. This backbone does not contain dCas9. 57,050 ...18,901 human genes along with 500 non-targeting controls Puromycin Doench , Root 92386 Human CRISPR inhibition...

...function under a wide range of highly controlled conditions in vitro , or, if you want to determine its ...Engineering Visualization and Tagging Purification Controlled Expression Reporter Plasmids Reporter Plasmids...Plasmids Tagging and Visualization Purification Controlled Expression Reporter Plasmids You may also like… ...produce large amounts of this protein and study it in vitro ? These, in addition to many questions about bacteria..., sometimes it’s best to delete genes from or introduce new genes and mutations to the bacterial genome...Manipulating the genome directly can give you more subtle control over protein expression and activity thereby limiting...Dammeyer Lactose/IPTG inducible vectors with LacIq, a stronger repressor than LacI that lowers basal protein ...

... undergo stringent quality control by Addgene or a trusted quality control partner before distribution...are initially titered by spectrophotometry using a NanoDrop spectrophotometer. If needed, the sample is...Addgene's antibody production methods and quality control processes for purified recombinant antibodies. ...production protocols, titering methods, and quality control are described below. Information for a specific...samples with >90% purity are distributed. Quality Control Each lot of antibody undergoes testing, either ...fluorescence pattern is compared to an untransfected control. Immunohistochemistry (IHC) Tissue sections are...Protein expression is compared to an untransfected control and the protein size is confirmed. Residual Plasmid...

... in search of inducible promoters capable of controlling eukaryotic gene expression. Several endogenous... regulator of gene expression, a tetracycline-controlled transactivator (tTA) was developed. tTA was created... transactivator rtTA ( r everse t etracycline-controlled t rans a ctivator) was created by fusing rTetR...and additional plasmids tetR-KRAB On Aebischer & Trono 16542 pBI-MCS-EGFP Expression of your gene of interest...Puro_Tet3G_3xFLAG_Twin_Strep Bidirectional promoter controls expression of gene of interest with Strep-Tag ...Blast (w756-1) Lentiviral reverse tetracycline-controlled transactivator 3 (rtTA3) expression vector, CMV... 3rd generation rtTA On Campeau 25434 pMA2640 Retroviral; CMV-driven; linked via IRES to EGFP-Blasticidin...

...facilitation, DNA amplification, and high-quality quality control measures such as titering, purity assessment, sterility...is used in a wide range of experiments, both in vitro and in vivo. It has low immunogenicity, is easy ...centrifugation purification Addgene’s comprehensive quality control process: Titering Purity assessment Sterility testing...produce the prep, and perform comprehensive quality control processes to make sure it is ready for your experiments...your AAV vector experience, from methods to troubleshooting insights, you make research more reproducible...gap between published methods and real-world troubleshooting. Eligible Plasmids The following plasmids are...including purification, titration, and quality control measures. We have distributed preps around the ...

...given donor-acceptor pair, the FRET efficiency strongly depends on the distance between the donor and ...linker-Cerulean-5aa linker-Venus-6aa linker-Venus V5V Anisotropy/brightness standard consisting of two Venus fluorescent...proteins connected via a 5 amino acid linker V17V Anisotropy/brightness standard consisting of two Venus fluorescent...proteins connected via a 17 amino acid linker V32V Anisotropy/brightness standard consisting of two Venus fluorescent...proteins connected via a 32 amino acid linker VVV Anisotropy/brightness standard consisting of three Venus... 5 and 6 amino acid linker, respectively VVVV Anisotropy/brightness standard consisting of four Venus ...5 and 6 amino acid linker, respectively VVVVV Anisotropy/brightness standard consisting of five Venus ...

...expression. γ-Retrovirus Plasmids | Guide γ-retroviruses, commonly referred to as retroviruses, are the most...integration. Community Resources Nolan Lab Retroviral Systems Trono Lab Lentivector Toolbox Guide to Generating...Adenoviruses are commonly used as vaccines because of the strong immunogenic response they induce. Some (oncolytic...this infectivity is limited to dividing cells. Retroviruses integrate permanently into the host genome, ...virus (as happens in nature), researchers can introduce plasmids to a host to generate virus. Furthermore...DNA >8 Kb 105 Dividing/Non-dividing No High γ-Retrovirus Stable RNA <8 Kb 80-130 Dividing Yes Moderate...

...Image from Fenno et al., 2014. INTRSECT INTRSECT (intronic recombinase sites enabling combinatorial targeting...and Flp) recognition sequences within synthetic introns. INTRSECT was first shown as a proof-of-concept...transgenic recombinase-expressing animal lines and retro-grade expressing viruses delivering additional recombinases.... Figure 2: INTRSECT works by inserting short, intronic sequences into the open reading frame (ORF) of...sequences (e.g. Lox sites, FRT sites) inside of the introns (A,D). The addition of these recombinase recognition...recognition sequences enables directional control of the portion of the ORF that is sandwiched between the... combinations of recombinases are present, the introns are excised during mRNA processing, producing a...

...Varies pBABE GFP - Retroviral gene expression pMKO.1 GFP - Retroviral shRNA... page . Retroviral Easy and safe to use pBabe plasmids - Mammalian retroviral gene ... retroviral shRNA expression MSCV-IRES-GFP - Mammalian retroviral gene expression...Tet-inducible lentiviral shRNA expression pMKO.1 puro - Retroviral shRNA expression pLenti CMV Puro... marker LZRS-Rfa or pMXs-gw - Mammalian retroviral Gateway cloning vectors Adenoviral...mammalian gene silencing pMKO.1 puro - Retroviral plasmid for mammalian gene silencing...

...provide a basic understanding of CRISPR biology, introduce the various applications of CRISPR, and help you...research. CRISPR Basics In this section, we will introduce the basic mechanisms of CRISPR and some of the...increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease activity SpRY...-occurring Acr (Anti-CRISPR) proteins can help control CRISPR activity. Acr proteins have evolved in phages...LmoCas9 and SpCas9. Acr proteins can be used to control both when and where genome editing happens. CASANOVA...Dominik Niopek’s lab , is a way to optogenetically control genome editing by fusing the anti-CRISPR protein...photosensitive LOV2 domain . Several labs have also controlled Acr activity with endogenous cell-type specific...

... Cut Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location...frameshifts, creating loss of function alleles. To introduce specific genomic changes, researchers use ssDNA...PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in DNA instead...mutants can also be used with a repair template to introduce specific edits via homology-directed repair (HDR...49410 pCFD3-dU6:3gRNA dU6:3 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid ...3tandemgRNAs dU6:1 and dU6:3 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid ...49408 pCFD1-dU6:1gRNA dU6:1 BbsI Injection or in vitro transcription Virmilion none, need Cas9 plasmid ...

... optimized dcas9 gene under the control of Murine Stem Cell retroVirus LTR promoter. The second plasmid...pgRNA_humanized ) contains a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom...as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Qi LS, Larson MH, Gilbert LA...vector (Leu2) that contains dCas9 fused to NLS controlled by TDH3 promoter 46921 pTDH3-dCas9-Mxi1 Yeast...and sgRNA targeting GAL4 UAS promoter (negative control) 46918 pU6-sgCD71-2 Human pSico-based U6 vector...Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TEF1...Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52 promoter, targeting endogenous TRE ...