...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...

... 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 ...

...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...

...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...

...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, ...

... 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...modifications can drastically 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... virus. In an AAV vector plasmid, they flank the gene of interest. A single ITR consists of palindromic...

...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 ...

...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 ...

...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 ...