We narrowed to 932 results for: Gene

...The white gene is most often used, but other selection markers include vermillion (another gene controlling...as species of gene. Find Gal4 and UAS plasmids at Addgene Choosing a vector to generate a transgenic fly...described the Gal4/UAS system used by geneticists to study gene function. In this final installment, I...you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated into...spot flies with the appropriate transgene. One such marker is the white gene, which restores eye color to...engineer new transgenic flies to study your favorite gene (YFG).  Sometimes, you want to use the Gal4/UAS...fairly straightforward. Basically, you place your gene of interest in a plasmid and get the vector incorporated...

...constant genes μ and ẟ. These are the first two genes in the heavy-chain constant domain gene cluster....constant domain genes, placing the variable domain genes next to a different constant domain gene. This process...Heavy chain constant genes are clustered together downstream of the variable region gene in this order: μ,...constant domain gene. For example if a B cell switches to producing IgE antibodies, the gene segments for...nomenclature. Antibody heavy chain proteins as well as the genes that encode those proteins are designated by the...μ, δ, γ, ε, and α. An antibody variable gene is initially expressed with the IgM and IgD constant domains...domains because they are the first constant domain genes in the cluster. However, when B cells switch to ...

... Acoustic reporter genes (ARGs) Acoustic Reporter Genes (ARGs) are engineered gene clusters that encode...for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference between...Resources on the Addgene Blog Learn more about in vivo imaging tools The luciferase reporter gene The green ...green fluorescent protein reporter gene Resources on Addgene.org Find plasmids for in vivo imaging with ... reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these limitations by using gas vesicle...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...

...numerous applications, including transgenesis, gene-trap screens, and gene editing. How does the piggyBac...levels of your gene of interest, as needed. Controlling the expression levels of your gene may be particularly...Transposagen.   How does piggyBac™ enhance gene editing? Gene editing can now be accomplished in a more... How does the piggyBac™ gene editing system compare with other methods of gene editing? There are primarily...Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing.,” Methods...remain, such as making gene edits completely seamless (i.e., so no other genetic alterations have occurred...cell lines and genetically-modified cell lines. Furthermore, the use of piggyBac™ for gene editing allows...

...any CRISPR-based gene editors, for correcting mutations or installing functional genes to study and potentially... number of genes with a single viral vector.   Fig. 1: Schematic of multiplex gene editing with...enable multiplex gene editing in a variety of cells and organisms. However, lengthy genetic payloads and ...demonstrating simultaneous editing of genes against multiple genetic diseases, such as muscular dystrophies...limited the scalability and efficiency of multiplex gene editing. Moreover, existing multiplex strategies...single array, has been a top choice for multiplex gene knockout or regulations, it lacks proper functions...derived tools are leading the edge of the precision gene editing. CRISPR-Cas9, the key component to enable...

...Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy. Curr Gene Ther, 13(6), 421–433...compartment, gene expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and...with the E1 and E3 (Early 1 and Early 3) genes deleted. These genes’ functions are rescued by helper plasmids... be expressed, since the early genes are absent, and the late genes (and their structural proteins, which... does not require de novo viral gene expression: Implications for gene therapy. Proc Natl Acad Sci U S...house a recombinant sequence (transgene) instead.    Although the late genes are usually present in the ...and E3 gene complementation. In target cells that are infected by the vector, only the transgene will be...

...hosts through horizontal (or “lateral”) gene transfer. Horizontal gene transfer can occur naturally through...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...Additional Resources on the Addgene Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: ...cell to another). Bacteria need special fertility genes to be capable of conjugation, and these are encoded...there is evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001...organisms to thrive in competitive environments. The genes encoding these functions form the basis of many ... Resistance (R) plasmids: microbial defense Genes allowing bacteria to survive toxins are the clearest...

... several genes spanning 33 cancer types. The types of mutations they identified included gene amplifications... plasmids each carrying a single gene with a defined mutation or gene fusion. Plasmids carrying the corresponding...how a given gene is involved in a complex pathway. Some of the most heavily-studied genes in cancer research...help cancer researchers studying an assortment of genes. Using patient data to identify mutations of unknown...corresponding wild-type genes were constructed as well. They called this library the CTD2 FASMIC Plasmid...thoroughly investigate any of the dozens of wild-type genes and their associated mutants in other model systems... Yiu Huen, et al. "Functional annotation of rare gene aberration drivers of pancreatic cancer." Nature...

...spatiotemporal expression of a gene of interest. This type of control over gene expression is possible in ...diseases that are caused by a specific gene. For example, several genes mutated in human cancers have a homolog... They share about 70% of their genes with humans and 85% of human genes associated with a disease have... share many genes with humans, are easily maintained in the lab, and have short generation times that ...Thus any genetic modification, such as expression of fluorescent protein or disruption of a gene, can be...Refinement of tools for targeted gene expression in Drosophila." Genetics 186.2 (2010): 735-755. PubMed ... of Drosophila research. Morgan discovered that genes were found within chromosomes using the fruit fly...

...Positive-selection vectors using the F plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841... experiments, and here we will focus on a unique gene that makes the popular GatewayTM method possible...accomplish this? Let’s start with a brief history of the gene and how molecular biologists have harnessed it to...cloning technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli,...and ultimately causing cell death. ccdA, another gene found in the ccd operon, codes for the antitoxin...vectors, called pKIL18 and pKIL19, contained the ccdB gene in frame with an MCS. E. coli that were transformed...transformed with the empty vectors expressed the ccdB gene and were therefore unable to propagate because CcdA...

...strategy is to knockdown a gene with RNAi. Here, a short sequence targeting the gene is cloned into a viral...whether it is an overexpression gene, downregulation via RNAi or gene editing/deletion using CRISPR. In...conjunction with a genetic manipulation so that behavior can be linked to a specific gene or brain region...viral vector should be used? Modifying gene expression Generally speaking, one may want to achieve either... application.  Genetic manipulation can also reduce expression levels of certain genes. A popular method...can now precisely control where and when certain genes are expressed to investigate their role in a specific...specific brain region. One option is to overexpress a gene that alters neuronal function directly such as receptor...

...platform. Gene Stacking As the name implies, gene stacking is the use of a combination of multiple genes to ...regulate the transcription of specific genes. What if one of those genes coded for a protein that regulated...combinations of different candidate genes at once to identify the best cocktail of genes when the relationship between...transcription of another gene? Now you have a genetic circuit! Genetic circuits are multigene systems whose protein...characterized activities with predictable behaviors. While a gene is the most obvious example of an individual part...transcribed together. They can express one or more gene(s), and typically begin with a promoter and end ...could be comprised of just three parts: promoter, gene, and terminator.     Fig 1. MoClo enables...

... the gene you would like to rescue with in the antisense orientation (let’s call the gene “GeneR”). The...rescue of a gene knockout: To test if the phenotype you are observing is really due to a single gene deleted...endogenous gene. Spatio-temporal expression of optogenetic reporters: Since most optogenetic reporters...transduced reporter gene. Generating a FLEx switch to control expression of an optogenetic reporter would ... were designed as a genetic tool for researchers to conditionally manipulate gene expression in vivo using...conditionally turn off one gene and activate another. In the field of mouse genetics, FLEx is currently being... vivo conditional gene rescue experiments, as well as for improvement of optogenetic adeno-associated ...

...recombination; ccdB gene for negative selection Used to clone attB-flanked genes of interest to generate entry clones...for recombination; ccdB gene for negative selection; elements to express the gene of interest in the appropriate...for gene expression.     The LR Reaction takes place between the attL sites of the generated entry...attB sites is generated. As in the BP reaction, a DNA fragment containing the ccdB gene is excised from... cloning to generate our desired constructs: lentiviral expression of the human KRAS gene in mammalian...   Pro Tip: Addgene also has ready-made entry clones available for many popular genes, including Hs....our website to search for your favorite gene! STEP 2: Generate an Expression Clone When making the expression...

...activation sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless...promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...reporter gene (3, 4), there are now many variations, including many that use auxotrophic rescue genes as reporters...Shapira S.K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and ...hybrid system (also called the interaction trap), genes encoding proteins of interest(POI’s) are fused to... in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing the target... the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact...

...approaches increase the size of the gene delivered by splitting the gene into two pieces: part A and part...packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. ...how do you fit a big gene into a tiny vector like AAV? By breaking the transgene into smaller pieces. ...acceptor sequence, the 3’ half of the transgene. The full length gene is reconstituted by either the overlapping...Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids. Human Gene Therapy...Chamberlain JS (2011) Gene Therapy of mdx Mice With Large Truncated Dystrophins Generated by Recombination ...retinal gene therapy Learn about the parts of an AAV transfer plasmid Resources on Addgene.org: Check...

...read-through transcripts of the effector gene. Effector genes, any gene that you want to control the expression...chemical inducers of gene expression (del Valle Rodríguez et al., 2011).  Genetics of SPARC Figure...Learn more about controlling gene expression in flies Resources on Addgene.org Find fluorescent protein...truncated attP sites to control recombination of the gene construct. A predictable number of cells will express...binds to the UAS binding motif, driving downstream gene expression. attP and attB, recombination sites. ...side of a stop cassette, upstream of an effector gene. In cells where the stop cassette is removed during...expression of either a stop cassette or an effector gene. HDVR (2X Hepatitis Delta Virus Ribozyme), a self-cleaving...

...typically include a gene that is advantageous for survival, such as an antibiotic resistance gene. In contrast...the target gene. Vital component for expression vectors: determines which cell types the gene is expressed...scientists use plasmids? Generally, scientists use plasmids to manipulate gene expression in target cells...replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing bacteria...MCS is often downstream from a promoter. Insert Gene, promoter or other DNA fragment cloned into the ...obtained. Selectable Marker The antibiotic resistance gene allows for selection in bacteria. However, many ...referred to as “vectors” or “constructs.” To insert a gene of interest into a vector, scientists may utilize...

...CRISPR-Cas9 constructs from Addgene to gene-edit mutations into XP genes and other genes involved in photosensitivity...researchers identified the gene responsible for the disease as ATP1A3. The gene encodes a sodium-potassium...Syndrome, a devastating genetic disease caused by mutations in the OCRL1 gene that leads to the early ...studies on OCRL1 already underway in his lab. The gene appears to play a role in many cell processes involving... learned there was a disease associated with the gene, it piqued their interest. They obtained cells from...McSweeney says they now think that mutations in the gene may prevent neurons from returning to basal conditions...Wattanasirichaigoon carried different mutations in a gene called SOX10. To understand the functional role ...

...between MARCH5 and WSB2 genes using Cas12a’s ability to knock out multiple genes in parallel. Lastly, this...to induce high levels of gene activation for both reporter and endogenous genes. It is highly specific ...cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation The Penn...10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation by: Gabrielle...many CRISPR-based genetic screening methods have primarily used Cas9 to knock out genes, researchers at ...multiple genes within a single cassette provides increased simplicity for combinatorial genetic screens...frankenbodies to amplify the signal.  Liu et al. Genes Cells. 2021. https://doi.org/10.1111/gtc.12893, ...

...demethylation Optogenetic repressors of gene expression in yeast Megan McClean’s lab generated an optogenetic...2019. Fusing epigenetic modifiers such as TET1 with inactive Cas enzymes can alter gene expression via...optogenetic toolkit that controls repression of gene expression in Saccharomyces cerevisiae. The system consists... enters the nucleus where dCas9-Mxi1 can repress gene expression. In the dark, the NLS is inaccessible...dilutions of CRISPRi in Caulobacter cells for ctrA gene expression using various dCas9. Image adapted from...takes several weeks to delete or deplete individual genes. Michael Laub’s lab developed a CRISPRi system to...downregulate expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived ...

...Entrez Gene matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list, ...correct Entrez Gene does not appear in the auto-populated list, try entering the gene's Official Full ...known as" aliases in the Gene/Insert box. Once you have selected the appropriate gene from the list, save ...bacterial strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any... for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should propagate... Encodes insert(s) A plasmid with one or more genes, protein coding sequences, or other elements inserted...Empty backbone A plasmid designed for cloning a gene/insert into it. Empty backbones often contain epitope...

...possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate gene expression) that ...large serine recombinase genes downstream of inducible promoters: the BxbI gene downstream of the anhydrotetracycline...the TP901 gene downstream of the arabinose (Ara)-inducible promoter (PBAD), and the A118 gene downstream...register to execute predictable gene regulation programs that specify which genes should be expressed or not... same gene regulation program. We recommend testing multiple register designs for the same gene regulation... paper. The paper also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved...promising strategy for linking RSM inputs to endogenous gene expression (2). Our paper (1) explains how to arrange...

... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...insertion of a reporter gene, or replacement of the murine sequence with a human gene. Advantages of using...conventional gene targeting. CRISPR tools were recently used to introduce a 25 kb human gene into the mouse...conventional gene targeting. It also costs less. With CRISPR, it may cost about $5,000 to generate founder ...for Tips on CRISPR Gene Editing in Mice Disadvantages of using CRISPR for generating mouse models Although...Jaenisch R (2013) One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated ...Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field. Mice have long been extremely useful...

...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... important influence on the expression of the transgene of interest and its localization in the membrane... tag protein can impact the expression of the transgene of interest. In our studies, mCherry interfered...

...is inserted at the 5’ end of a gene, the SEC separates the targeted gene from its promoter, creating a ...the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...animals per gene, they obtained the desired insertions on the first attempt for 6/7 genes. The data are...hands-on time. Creating a modular, selection-based gene tagging approach Multiple features of C. elegans...uses a selection cassette containing genes for hygromycin resistance and the Roller movement phenotype. To...design and clone 500-700 bp homology arms for your gene of interest into the FP-SEC vector using Gibson ...loss-of-function allele as well as a promoter-FP fusion for the gene of interest. L1/L2 larvae from these strains are...

...sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018   pdDronpa Cas9, dCas9 Gene editing, transcription...showing that this genetically encoded, photocaged Cas9 system could silence endogenous gene expression - demonstrating...photocaged-gRNA Gene editing For more details, check out this comprehensive review comparing optogenetic CRISPR...a system that would use light to turn on and off gene expression while imparting spatiotemporal control...and move the activator into position to activate gene transcription. The researchers tested a variety ...researchers showed that expression of the human ASCL1 gene could be spatially controlled in a reversible and... to develop an optimized photoactivatable CRISPR gene activation system, but settled on a different optimal...

...library that has 10 gRNAs per gene and targets all ~20,500 protein-coding genes. Unique, non-overlapping sites... identified ricin toxicity genes. The screen also discovered several genes previously not associated with...p-values of known ricin regulation genes compared to results generated using non-targeting guides as controls...-throughput systematic approach for identifying genes involved in a biological process. These screens ...toxicity screen since essential and non-essential genes for both pathways had previously been identified...identifying >88% of previously identified essential genes while previous Cas9 and shRNA library screens only...only identified  60% of the essential genes. The ricin toxicity screen had a 10% false discovery rate ...

...et al., Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in... AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot replicate on their ...et al. "Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in...applications and gene therapy: they can be produced at high titers, can accommodate large transgenes, transduce...recombination-competent adenovirus (RCA) can be generated if the E1 gene from the packaging cell line is transferred...for the generation of RCA and reduced immunogenicity. In addition to E1/E3, non-structural genes E2 and...proteins in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning...

... you started. Any given gene’s webpage will have information on where the gene is expressed within the...from mutations in the gene, and whether any publications have mentioned the gene. The Bloomington Drosophila...also has collections of flies for in vivo gene knock down and gene expression. In the next post, you’ll ...favorite gene (YFG) in Drosophila (and even if there’s not!) the wealth of Drosophila genetic tools available...PhD student at Harvard University. Do you have a gene of interest but have run into a wall trying to study...it? It happens. Is it an evolutionarily conserved gene? Can you find an ortholog in the Drosophila genome...a chance of seeing a phenotype since additional genes that can do the same function are less likely to...

...nearby genes and its role in gene expression, though this may still unexpectedly affect nearby genes (Figure...protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe..., or permanently disable, protein-coding genes by generating insertions or deletions in the DNA sequence...only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest of our...manipulate and study in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined...multiple gRNAs poses the risk of disrupting adjacent genes, especially those regulated by or overlapping with...). The interactions between ncRNAs and adjacent genes are complex and not yet fully understood, so it ...

... cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...sgRNA-targeted gene, resulting in increased activation of endogenous transcription of the target gene. The brilliant...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant*...resistance gene (missing the first two codons). Two stop codons are inserted between the truncated gene and ... inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (...on target gene activation. The novel dCas9-VPR activator is capable of enhancing target gene expression...

...several candidate inhibitors of gene X in a screen. You generate a gene X knockout in a cancerous background...inhibit, activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...some of the candidate genes! Disease model generation CRISPR has made the generation of genome edits accessible.... These hits can reveal individual genes or sometimes even genetic pathways that are worth investigating...known – repeat expansion, gene knock out, etc. – CRISPR can be used to generate cell line or animal models...approaches. The first route involves identifying a gene/pathway to develop a drug for (aka a druggable target...straightforward causes (a specific mutation in a known gene = disease onset) may be best served by this type...

... popular tool for gene delivery, but it has a drawback: how do you ensure your gene goes where you want...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...projections. Generating animal models: Transgenic animals allow for the expression of genes in particular...Additional resources on the Addgene blog Read more about using AAV for retinal gene therapy Find more about...want it to? Knowing that a gene is expressed in a particular cell type is important not only for translational...translational research, such as gene therapy, but also basic research. To improve cell-type specificity of... transcription initiation sites of at least two genes with high expression and high cell-type specificity...

... after light stimulation. Under those optogenetic controls, gene expression levels reliably increased ...could be by applying it to altering gene expression via epigenetics with the addition of histone modifiers...conventional optogenetic tools." In total, the researchers applied the LITE system to 28 different genes to establish...transcriptional effectors (LITEs) designed to bind specific genes and turn them on or off, literally at the flip of...are able to react and learn. All of this requires genes to be regulated dynamically.” To understand how ... tools to manipulate the expression of specific genes in the brain in reversible fashion. And that’s exactly...demonstrated use of the LITEs to control gene expression in mouse neurons and in the brains of living mice ...

...you are trying to do: gene knockout, a specific base edit, or modulation of gene expression. Location ...Created with BioRender.com. The hammer: Gene knockout by NHEJ Gene knockout with CRISPR technology is usually...multiple sequences targeting a gene to score in order to call that gene a hit.  This is an exciting time...optimize numerous parameters to generate the desired edit. The wrench: Gene activation and inhibition by ...edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post...indels, it's not so important what location in the gene you target, but it is important that your gRNA sequence...creating a non-functional allele. For a 1 kilobase gene, since potential target sites occur ~1 in every ...

...acid sequence from within Snapgene, we can confirm the identity of the gene and check for any mutations...we identify the gene, we can easily annotate it by making a custom feature in Snapgene. This saves us ...frames, which is often our first clue about where the gene of interest might be located. By easily using BLAST...plasmids. We’ve also talked about how our new Snapgene generated maps provide improved feature detection with...’ll walk you through how a scientist at Addgene uses Snapgene to confirm the sequence of a plasmid and...through our Snapgene powered maps and sequence analysis tools.     The main ways we use SnapGene for plasmid...version of Snapgene to visualize the sequence and look for features. The first thing that the Snapgene software...

...Strategies to Make Gene Therapies More General (more than correcting a defect in a single gene) 26:00-28:39 ...39 : Methods for Delivering Multiple Genes to Cells for Gene Therapy 28:40-35:10 : GFP Activatable Tools...Targeting in Retinal Gene Therapies 10:17-14:34 : Progress in Treating Macular Degeneration and Other Retinopathies...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed... gene therapy...Production Specialist at Addgene. He is the audio engineer and producer for the Addgene Podcast.     References...plasmids from this publication at Addgene. Resources on the Addgene Blog Read our Recent Blog Post on...

... epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein super family). ...knock-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first ...designed for ~250 genes, we have had about 30% tagging success, although many of these genes were expressed...facilitated easy generation of clonal knock-in lines that we now use for IP and ChIP assays. Tag 96 genes at a time...Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian .... Additional Resources on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids...Fluorescent tagging of endogenous genes with SapTrap Resources on Addgene.org Browse CRISPR plasmids Check...

... with ITRs that flank the gene cassette, which contains a promoter, the gene of interest, and a polyA ... DNA molecule as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs...vectors, modifications can affect gene transfer efficiency and transgene expression (Zhou et al. 2017). ...Terminal Repeat Sequence Role in Vector Transgene Expression. Hum Gene Ther, 31(3–4), 151–162. https://doi.org...biological tool that allow scientists to express a gene of interest in a cell or animal model. AAV plasmids...ITRs, and the ITRs are retained with the gene in a recombinant AAV (rAAV) vector (Figure 1). Everything...plasmids. In an AAV vector plasmid, they flank the gene of interest. A single ITR consists of palindromic...

...an unpublished set of Zika gene plasmids to Addgene’s repository. The genes, from the Zika virus Asian...vectors targeting their genes of interest to generate mAID fusions to endogenous genes. Cell lines with the...targeting a total of 20,121 genes within the human genome (the identity of the genes and target sequences can...expression level for their gene of interest. Although the parts involved in altering gene expression are relatively...contains ~90,000 gRNAs that target ~15,000 genes with 6 gRNAs per gene. The second is a "supplementary" library...library to generate a population of HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA... greatly simplified the process of generating multiple E. coli gene expression plasmids with the Cross-disciplinary...

...been used for many years for in vivo delivery and gene therapy and represent one of the most studied viruses...CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted DNA knock-in (right...binds to target DNA. In the final step of targeted gene knock-out (left), a fragment is deleted from two... This capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral...Joining (NHEJ) DNA repair mechanism to knock out genes as a result of an insertion, a deletion or a frameshift...with Cas9 and a gRNA designed to target a mutated gene. In a Scientific Reports paper introducing the delivery... CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in the various cell types...

...efforts to regulate gene expression were limited to inducible promoters or direct gene knockout. In contrast... of gene expression. They successfully knock down expression of essential and non essential genes in a.... These could be genes, specific polymorphisms, including antibiotic resistance genes. This approach successfully...resistance pCRISPR: carries a spacer targeting the gene of interest and kanamycin resistance E. coli carrying...wild-type sequence persists, Cas9 will cleave the gene of interest, inducing a lethal DSB.     This system.... pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific gRNA(... cleavage induces HDR to directly modify a given gene. CRISPR-tranposons By combining CRISPR editing and...

...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https...Specificity of gfaABC1D-Driven Transgene Expression in AAV-Mediated Gene Transfer. Molecular Therapy - ...easier to do the validation first.  AAV-mediated gene expression can differ extensively depending on the... cells in far away brain regions expressing your gene of interest. This can cause issues in tracing studies...can occur. Promoters The promoter controls how the gene of interest will be expressed; it can be ubiquitous...chicken-beta-actin or CAG can drive strong early gene expression, but can become methylated over time,...tyrosine-hydroxylase (TH), can limit which cells express your gene. Off-target expression can still occur (Fig. 3) ...

...this plasmid with your gene of interest, you would replace mCherry with your gene of interest. pBLADEONLY...already have a pBAD33-based plasmid expressing your gene of interest, all you have to do is co-transform ...co-transform pBLADEONLY. That way, a previously cloned gene of interest, under the pBAD promoter, can be controlled... Resources on Addgene.org Check out the Molecular Biology Reference Download Addgene's Plasmids 101 eBook...simply more precise. “We show that you have less heterogeneity in the population and you can do all of these...permission.   BLADE is just one of the several optogenetic tools that Di Ventura’s lab and Khammash's lab...lab have created and shared with Addgene. You can find all the plasmids deposited from Di Ventura's lab...

...using AAV as a tool for gene expression in mammals. Resources on Addgene.org Search for AAV vectors ...One of the biggest barriers to gene therapy treatment of central nervous system (CNS) diseases is the...has not been identified. While LY6A or other Ly6 gene family members are linked to viral susceptibility...large phenotypic effects such as those that affected gene expression, mRNA splicing, or protein coding regions...as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive phenotype...permissiveness. The Wilson Lab also looked for the gene variant responsible for the PHP.B permissive phenotype...inheritance pattern that suggested there was a single gene that had two codominant versions or alleles that...

...while G418 (geneticin) is effective at inhibiting the eukaryotic ribosome. One resistance gene on your vector...used in the lab contain an antibiotic resistance gene to allow for antibiotic selection, but these resistance...aspect of ribosomal function. A puromycin resistance gene on your vector can protect both plasmid-carrying...block protein synthesis, and the neomycin resistance gene protects against any of them. But different aminoglycosides...cause mutations even in cells with the resistance gene, so it isn’t a good fit for all experiments. And...experiment should still include an antibiotic resistance gene for growth in bacteria. Expression You’re almost...systems. But will your cells actually express your gene of interest? Many promoters are species-specific...

...cost-effective manipulation of gene expression in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https...system, in which the required adenoviral helper genes are supplied on two different packaging plasmids...pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing replication ...replication factors and serotype-specific capsid genes.  In this helper-free system, HEK293T cells, expressing Ad...transfected with your transfer plasmid, carrying your gene of interest, and the two helper plasmids, commonly...derived inverted terminal repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org...genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...

...the Grousbeck Family Chair in Gene Therapy, Director of the Grousbeck Gene Therapy Center, Associate Director...nonprofit companies. His lab focuses on gene therapy work for genetic diseases and over the past few months...deposited AAV constructs for gene delivery. In 2018, he interviewed Addgene co-founder Melina Fan and former...PhD Khandan Baradaran’s expertise in viral safety, gene therapy vector development, vaccine development,...Dimension Therapeutics, she helped build and grow the gene therapy division of the company.  Ultragenyx’s goal... systematically test the function of all 20,000 genes in disease models and then focus drug discovery ...to crises like those and ended up in the field of gene therapy.” He’s also worked on vaccines throughout...

...scRNA-seq), now allows scientists to identify the gene expression patterns unique to the few cells that...reprogram. Unfortunately scRNA-seq only captures the gene expression of cells at a single time point, making...at the single-cell level, thus linking ancestral gene expression patterns with reprogramming outcomes ...constructing lineage trees and comparing them with the gene expression changes of more than 100,000 cells undergoing...partially reprogrammed and re-expressed fibroblast genes. Most cells ended up taking the ‘dead-end’ path....cells with shared CellTagD13 tags often had similar gene expression pattern changes and reprogramming outcomes...tracing experiment, the Morris lab identified a novel gene, Mettl7a1, whose expression helped coax cells towards...