...Pre-constructed pairs of TALEN plasmids targeting zebrafish genes....target sites within various endogenous zebrafish genes; many of these were generated as part of an NIH-...TGCCTGGCACAGGGGCTctccaccatgctggccAACCTGTTCTCAATGA kinesin-family-member-13a TAL3106 & TAL3107 TGTTCTCTGTTTGAGCGAgtctccacacagcagaGCGACAGTAACAGCTTCA...

...in Mammalian Cell Lines CRISPR: Protocol for Genomic Deletions in Mammalian Cell Lines Other Addgene Resources...loss-of-function studies of genes and genetic elements in mammalian cell lines. Protocol For referenced ...CRISPR/Cas9 Clones for Deletions and Clone Selection For suspension cells, transfer all clones to a single...Validation of Biallelic Deletion Clones In order to characterize obtained clones and validate a successful knockout...JoVE) about genomic deletions in mammalian cell lines using CRISPR/Cas9 CRISPR...Cas9 to create genomic deletions in mammalian cell lines. Along with the video, you can find the protocol...Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9. Bauer DE, Canver MC, Orkin SH. ...

...Fluorescent Protein Guide Empty Backbones Fluorescent Protein Guide: Empty Backbones More Fluorescent Protein...Addgene has assembled a collection of empty plasmid backbones with different fluorescent tags for you to create...generated with the help of Erik Snapp . Empty Backbones for Fluorescent Protein Fusions (Organized by ...Excitation and Emission maximum are listed in nm. Brightness is the product of exctinction coefficient and...Blue/UV Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Sirius 355 ...Top Cyan Protein Excitation (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Aquamarine ...

... Collections Empty Backbones Choosing Your Perfect Plasmid Backbone Empty backbones are plasmids containing...A guide to Addgene's empty vector backbones curated by species, epitope tags/fusion protein, selectable...guide to a selection of Addgene's empty vector backbones. For the most part, we will assume that you want... Host Relevant Promoters Representative Empty Backbones Mammalian CMV, SV40, EF1a, CAG, Ubc Transient ... Tet-inducible Find more Tet-inducible empty backbones on our Tetracycline (Tet) Inducible Expression...Hygromiycin resistance marker Also see more empty backbones on our Plant Plasmids and Resources collection...Protein Common uses Representative Empty Backbones Flag Epitope tag c-Flag pcDNA3 or FLAG-HA-pcDNA3.1...

...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m in neurons Yulong Li 123308-AAVrg...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m in neurons Yulong Li 123309-AAV9...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h in neurons Yulong Li 123309-AAVrg...the genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h in neurons Yulong Li 123310-AAV9...the genetically-encoded fluorescent norepinephrine(NE) control sensor GRAB_NEmut in neurons Yulong Li 123310...the genetically-encoded fluorescent norepinephrine(NE) control sensor GRAB_NEmut in neurons Yulong Li 125560...combination with Cre-dependent transgenic mouse lines or co-infection with Cre dependent viral vectors...

...Syn genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1m Norepinephrine sensor Li 123309...Syn genetically-encoded fluorescent norepinephrine(NE) sensor GRAB_NE1h Norepinephrine sensor Li 123310...control norepinephrine sensor (does not respond to NE) Norepinephrine sensor Li 20297 pAAV-EF1a-double ...serotype to deliver Cre-dependent transgenes in Cre transgenic lines. As part of our standard viral production...nuclear dTomato Calcium sensor Ting 100853 pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 Syn Cre-dependent jRGECO1a...jRGECO1a Calcium Sensor Kim , GENIE 100854 pAAV.Syn.NES-jRGECO1a.WPRE.SV40 Syn jRGECO1a Calcium Sensor Kim...

...pcDNA3.1-hSNCA-NE SNCA NE CMV Parkinson's Shu Leong Ho 102363 pcDNA3.1-hNurr1-NE NR4A2 NE CMV Parkinson's...pSIN-PAmCherry-mSNCA-NE SNCA NE EF-1 alpha Parkinson's Shu Leong Ho 102366 pSIN-hSNCA-NE SNCA NE EF-1 alpha Parkinson's...215486 FUW-hSNCA-NE SNCA NE Ubc Parkinson's Ophir Shalem 215487 FUW-hSNCA(D2R)-NE SNCA NE Ubc Parkinson'...Alzheimer's Hélène Marie 107544 pAAV-AICD-NES-IRES-hrGFP APP V5 CMV Alzheimer's Hélène Marie 107548 pAAV-syn-AICD-IRES-hrGFP...Parkinson's Ophir Shalem 215488 FUW-hSNCA(D2E)-NE SNCA NE Ubc Parkinson's Ophir Shalem 215489 FUW-hSNCA-GFP SNCA...pAAV-syn-AICD-IRES-hrGFP APP hSyn1 Alzheimer's Hélène Marie 107594 pCS2-sod1 SOD1 SP6 ALS Qing Deng 107798...SFFV Parkinson's Denes Hnisz 145270 pHR-TBP(53Q)-IDR-mCherry-Cry2-NLS TBP Parkinson's Denes Hnisz 145283 ...

...knockout screening in human cells. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods . 2014 Aug;11(8):...contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming...CRISPR-mediated nucleic acid cleavage. We have harnessed the type II CRISPR nuclease system to facilitate...implemented in mammalian cells by co-expressing the S. pyogenes Cas9 (SpCas9) nuclease along with the guide RNA...for the transcriptional activation of endogenous genes. It consists of three components: A nucleolytically..., Nature 2015). The dual vector system uses S. pyogenes Cas9 (SpCas9), using one vector to express SpCas9...

...SF-iGluSnFr Glucose Sensors iGlucoSnFr Norepinephrine (NE) Sensors GRAB_NE nLight Oxytocin (OT) Sensors GRAB_OT...iGlucoSnFr mRuby2 Constitutive 9 Looger Norepinephrine (NE) Sensors 123308 pAAV-hSyn-GRAB_NE1m Syn GRAB_NE1m... Griesbeck Calcium Sensor: jRCaMP1 100846 pAAV.CAG.Flex.NES-jRCaMP1a.WPRE.SV40 CAG jRCaMP1a none Cre dependent... dependent 1 Kim , GENIE 100848 pAAV.Syn.NES.jRCaMP1a.WPRE.SV40 Syn jRCaMP1a none Constitutive 1, 9 Kim...Kim , GENIE 100849 pAAV.CAG.Flex.NES-jRCaMP1b.WPRE.SV40 CAG jRCaMP1b none Cre dependent 1 Kim , GENIE ...GENIE 100850 pAAV.Syn.Flex.NES-jRCaMP1b.WPRE.SV40 Syn jRCaMP1b none Cre dependent 1 Kim , GENIE 100851 pAAV.Syn.NES-jRCaMP1b.WPRE.SV40...CaMPARI none Constitutive 1, 5, 9 Looger 101060 pAAV_hsyn_NES-his-CaMPARI2-WPRE-SV40 Syn CaMPARI2 his Constitutive...

...AID) enzyme. AID, however, only changes cytidines or guanines to the other three bases, which limits the...directed mutagenesis to generate specific mutations in your plasmids Use REPLACER Mutagenesis to mutate...Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes...evolution of new phenotypes, and forward genetics harnesses this at a large scale by screening diverse libraries...region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods...error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli, but this approach requires special...

...practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene depositor...with Venus. This luminescent technology is based on the principle of bioluminescence resonance energy ...reporter genes for endogenous protein fusions created using CRISPR/Cas9. eNLs offer high luminescent signal... a few drawbacks associated with luminescent imaging: A luminescent substrate is required for imaging...Researchers have long been interested in using luminescence to get around these issues, but this solution...fusion proteins. Solving the problems of low luminescence By itself, Renilla luciferase has a low quantum... hasn’t stopped some scientists from using chemiluminescent proteins for imaging, but it has precluded...

... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...Laursen from Cornell University. Site directed mutagenesis is a highly versatile technique that can be used...analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG...Cas9 induced cleavage. Summary of Site Directed Mutagenesis In brief, point-mutations can be introduced to...the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...

...expression of surrounding genes. However, there are ways to create scarless mutations, ones that don’t leave any...toxins for counter-selection In 2015, the Chen lab harnessed a group of bacterial toxins to facilitate efficient...Red that contained one of eight bacterial toxin genes isolated from E. coli or Psuedomonas aeruginosa....toxins, and phage holins. They hypothesized that genes that had evolved specifically for toxicity would... pathogenic Proteobacteria. We placed the toxin genes under control of a rhamnose-inducible promoter, ... they are just spontaneous antibiotic resistant clones. But if they are blue or magenta, they are real...

...target-specific ones to application-specific ones to anything goes. We've shared the ones we're familiar...search engines and antibody data repositories to find an antibody for your experiment. Search engines let...which share validation data, and antibody search engines, which allow you to search many different antibody...Table 1: Antibody data repositories and search engines Website Targets Application Purpose ...

...allowed scientists to tag and light up endogenous genes of interest to better understand normal protein ...vectors. These vectors can be used to tag endogenous genes and simultaneously introduce a selection marker ...vectors can be easily modified to tag hundreds of genes, generating powerful genetic screening libraries...easily create C-terminal tag fusions of endogenous genes in human cells. To use CRISPaint, users need 3 vectors... Learn more about these constructs and the cell lines they’ve been used to create in the Allen Institute...

...would spend hours to days trying find their correct clones. Additionally, the presence of a plasmid is disadvantageous...Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scientists introduce an...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8...Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You may also...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...transient transfection approaches, generating cell lines using lentiviral vectors enables long-term protein...293T cells but can be adapted to alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells...needed Day 14–18: Expand and harvest stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet...

...public, not private interests. On this nonprofit awareness day, we layout the ways in which we promote our... nonprofit awareness day...

...Healthcare consulting firms that specialize in business strategy tend to be mostly engaged by pharma and...companies. Medical Affairs groups work on building awareness among physicians and scientists about clinical...collaborative, and financial functions associated with the business. Commercial teams routinely work with consulting... a very short amount of time. Such a decision determines whether the client invests substantial monetary...consultants are usually trained in the life sciences, in business, in healthcare economics, or in some combination... you, you’ll have the opportunity to learn the business side of consulting cases, while feeling like you...healthcare space that can benefit from the blend of business and scientific understanding of successful therapeutics...

...assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to...REPLACR-mutagenesis could save busy scientists a lot of time. Let us know how REPLACR-mutagenesis works ...Comparison of REPLACR-mutagenesis with other common methods for site directed mutagenesis   References 1. ...al. (2016). “REPLACR-mutagenesis: a one-step method for site-directed mutagenesis by recombineering.” ...Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular... Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on in vitro ...structure or too much sequence similarity. REPLACR-mutagenesis: Letting bacteria do the work If you’ve conducted...

...Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013)...

...yeast gene of interest. These fluorescently tagged genes are expressed under native conditions and allow ...looking at their performance in categories such as brightness, stability, and disruption of the tagged protein...be interested in attaching epitope tags to their genes of interest, allowing for easy capture and detection...

...temperature ranges and make them grow better at suboptimal ones, which could allow even your regular E. coli to ...where we could incorporate cold-tolerance inducing genes within a regular mesophilic host system. This would...folding at low temperatures, since mesophilic chaperones would not function well under suboptimal conditions...engineering a plasmid which contained cold tolerant chaperones (the Cpn 10/60 isolated from Oleispira antarctica...the figure below shows, E. coli expressing the chaperones protein resulted in higher levels of growth compared...

...effects. One-pot saturation mutagenesis The one-pot saturation mutagenesis technique described in Wrenbeck...saturation mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How .... “Random Mutagenesis by Error-Prone PCR”. Methods in Molecular Biology In Vitro Mutagenesis Protocols...Efficient, Expansive, User-Defined Mutagenesis.” Ed. D. Dafydd Jones. PLoS ONE 7.12 (2012): e52031. PubMed...the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used...library generation prior to one pot saturation mutagenesis Several methods can be used to create mutational... shortcomings compared to one pot saturation mutagenesis. Error-prone PCR: This method amplifies the...

...website navigation paths, the organization and completeness of our information, and the accuracy and relevance...

...bound to modified proteins called histones. We can detect modified histones with “GPS for proteins,” ChIP-... of modified histones, a genomic region that has mono-methylated and acetylated histones, but not tri-...which NANOG-related genes are critical to stem cell survival. Knowing which genes influence the survival...Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome ...tri-methylated histones, functions as an enhancer. We can therefore denote enhancers as follows: (mono_methylation...combination of coiled DNA and structural proteins called histones. Chromatin folds into secondary structures (loops...

...Bacterial TAG Ryan Mehl 174081 pAcBac1-NES-Flag-R2-84-MbRS Tetrazine3.0 NES-Flag-R2-84 tRNA synthetase M. barkeri...growth medium, ncAA concentration, and the cell lines used previously. Remember that the orthogonal pairs...aminoacylate only the orthogonal tRNA, and not endogenous ones. Endogenous synthetases cannot aminoacylate the ...mazei Mammalian TGA Simon Elsaesser 174901 pAS_MmaPylT_EF1_NES-MmaPylRS WT PylRS M. mazei Mammalian TAG ...TAG Simon Elsaesser 174902 pAS_MmaPylT_EF1_NES-MmaPylRS AF PylRS (Y306A, Y384F) M. mazei Mammalian TAG Simon...Thomas Huber 182287 pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF) Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA synthetase...TAA Wenshe Liu 182653 pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF)_IRES_eRF1(E55D)-HA Y306A/Y384F (AF) pyrrolysine...

...coming from.   Acoustic reporter genes (ARGs) Acoustic Reporter Genes (ARGs) are engineered gene clusters... reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these limitations by using gas vesicle... require injection of substrate to produce bioluminescence. Substrate pharmacokinetics and biodistribution...localization: In vivo imaging with fluorescent or bioluminescent reporters usually only detects the signal of...and high spatial resolution. By piecing together genes from gas vesicle operons from two different bacteria...their ultrasound signal. Using acoustic reporter genes for in vivo imaging Following in vitro characterization...S.P., & Shapiro, M.G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian...

...exploding. Researchers are increasingly able to harness the body’s immune system to fight progressing cancers...this multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting...

...are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used to study...Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when expressed, enable cells... vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. That is,...gas vesicle-expressing species. The gas vesicle genes from Serratia sp. 39006 produced the highest levels...bARGSer expression, from left to right: colony whiteness on inducer plates, white inclusions in cells with...cell line with strong gas vesicle expression and robustness without chromatin-opening agents. It ended up...being very straightforward to engineer various cell lines from different tissues and mammalian species to ...

...Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized ...Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations ... and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our...targeted deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given...cells can be easily identified. Even in the case of clones with monoallelic deletion, the nondeleted allele...validation, and screening by PCR for biallelic deletion clones:    Like the video? The Orkin and Bauer lab's...enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 2013 Oct 11;342...

...immortalize CDG patient-derived immune cell lines and use these lines to study how immune processes and biological...research on diseases of glycosylation, arginine mutagenesis, and neural development. Paula Videira: Congenital...studying the function of the mouse Nfia, Nfib and Nfix genes in brain development since 2000 and have shown several...

...international researcher who uses plasmids containing genes from rabies and vesicular stomatitis virus, you ... export control exceptions for rabies and VSV-G genes, plasmids ship out approximately three weeks faster...

... our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward...our engineered devices, recombinase-based state machines for your own experimental needs. For more context...timing of these factors. Recombinase-based state machines (RSMs), which take on a particular DNA-sequence... from different recombinases are overlapping or nested on a register, then the operation of one recombinase...replication), contains large serine recombinase genes downstream of inducible promoters: the BxbI gene...The paper also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved ...predictable gene regulation programs that specify which genes should be expressed or not expressed in each state...

...localization signal (bNLS) and a nuclear export signal (NES), both which are phosphorylatable. Prior to phosphorylation... phosphorylation bNLS activity is suppressed and NES activity is enhanced, leading to cytoplasmic translocation...of the phosphorylatable residues and the bNLS and NES sequences, which have previously been characterized.... for more details on the design of the bNLS and NES sequences. Disadvantages of KTRs KTRs also have some...

... fragmented genomes is the key to fitting large genes into AAV First, some history. In 2008, the Auricchio...mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines the overlapping and trans-splicing methods. The... A (2008) Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective...Chamberlain K, Riyad JM, Weber T (2016) Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated... Auricchio A (2013) Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med ...

...repress the expression of genes primarily involved in development. These genes are known to play a role...many contexts within the fly including oogenesis and cytokinesis. Why name this protein after a vegetable..., two genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important...bagpipe are both homeobox genes that are part of a genetic cascade that determines the cell fate of the dorsal...two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila." Genes & Development...they can name a gene and many of the interesting ones come from the fly community. Not surprising, this... our repository that you can use to study these genes! Groucho, a transcriptional co-repressor The first...

...2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian...through a host organism, the expression of some genes can change depending on the location. Current methods...of microbes rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue...vivo using ultrasound. These ARG plasmids contain genes for “gas vesicles,” a gas-filled protein structure...demonstrates engineered gas vesicles as the first reporter genes using ultrasound and provide a major benefit to ...active miRNA’s unique to HEK293FT and HeLa cell lines. When the miRNA activity profile for a given cell...segment Understanding the connection between specific genes and an immune response has been a longstanding challenge...

.... 25(10):1581-9. PMID: 26355004 Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl... anti-CRISPR genes in bacteriophages infecting Pseudomonas aeruginosa . Anti-CRISPR genes employ varied...system - utilizing various CRISPR-associated (Cas) genes to not only store a record of invading phages but...Cas proteins. Makarova et al. ’s classification defines 5 types and 16 subtypes based on shared characteristics...genomic DNA. Type II CRISPR was the first system harnessed for genome engineering, with Type V following ...Csf1) Type VI (Cas13) Fighting Back: Anti-CRISPR Genes in Phage The CRISPR adaptive immune system seems.... Pooled gRNA libraries can be used to identify genes that are important to a given phenotype. Current...