...relies on the sacB gene product of B. subtilis (Hmelo et al., 2015). The sacB gene product is a sucrase... in the chromosome is to disrupt expression of a gene with an antibiotic resistance cassette. This leaves...sometimes interfering with expression of surrounding genes. However, there are ways to create scarless mutations...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, .... "Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria." Plasmid 51.3 (2004...

...particular gene mutation, southern blotting can be used to identify the presence of that mutated gene. In southern...the location of genes. With this knowledge, researchers were able to target specific genes for deletion ...southern blotting being used to identify a gene mutation is the genetic test used for sickle cell anemia (2)...probe (for example, has the sequence of the mutated gene the researcher is searching for), then the probe...to identify the presence of the mutated β globin gene that causes sickle cell anemia. The mutation happens...questions. Real-time PCR detects and quantifies a gene or DNA sequence of interest by recording DNA abundance...fluorescence in situ hybridization for detection of gene copy number changes." Protocols for Nucleic Acid...

...investigation into the regulation of your favorite gene. But have no fear - I’m here to introduce you to...genome – could be affected, leading to unpredicted gene expression changes (Sapozhnikov and Szyf 2021, Charlton...interact with transcriptional machinery to modify local gene expression, confounding any potential conclusions...causal relationship between DNA demethylation and gene expression (Sapozhnikov and Szyf 2021).    ... expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy editing...Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts...Methylcytosine Hydroxylation, Active DNA Demethylation, Gene Transcription, and Memory Formation. Neuron 79, ...

...., 2018). Hybridomas can lose gene expression of the mAb-encoding genes or fail to revive after cryopreservation...monoclonal antibodies that are generated in vitro using synthetic genes that are typically expressed from... integrated sequence in a stable cell line. The genes encode the heavy chain and light chain for the antibody...Figure 2: (Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid. (Right...better and better at identifying and cloning the genes for antibody expression. If starting from a protein...sequences of the antibodies and then synthesize the genes that encode those amino acids (Tran et al., 2016... Antibody? animation Resources on Addgene.org Check out Addgene's antibody plasmid collection ...

... insert the FP gene into the target gene’s native locus, but introducing large genetic changes in C. elegans...glycosyltransferase genes and other proteins related to cellular glycosylation. Get the library at Addgene CasRx ...cassette exons and disrupts genes with alternative exons. Get the library from Addgene New from the viral...library inhibits over 18,000 human genes. Find the lentiviral preps at Addgene hSyn1-SIO-stGtACR1-FusionRed...So how can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach. Split-wrmScarlet... validated this tool by targeting six different genes whose protein products have distinct localizations... number, generally very small, and can contain different neurotransmitters. Using genetically encoded ...

...particles. But, exo-AAVs may open new ways of improving gene delivery. They increase the efficiency of transduction...for shielding neutralizing antibodies and robust gene delivery in vivo." Biomaterials 35.26 (2014): 7598...PMC4104587. György, Bence, et al. "Rescue of hearing by gene delivery to inner-ear hair cells using exosome-associated.... "Microvesicle-associated AAV vector as a novel gene delivery system." Molecular Therapy 20.5 (2012):...PMC3345986. Meliani, Amine, et al. "Enhanced liver gene transfer and evasion of preexisting humoral immunity... "Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...plasmids from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli...optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...used to engineer new pathways with sophisticated gene expression controls. Most cell types do not respond... can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive two-component... was only about 10-fold, precluding the study of gene expression at very high or very low levels. Schmidl...researchers to better fine-tune the level of output gene expression based on the input light intensity. In...the plasmids from this publication at Addgene. A synthetic genetic edge detection program. Tabor JJ, Salis...

...lines and are not used for general plasmid selection. Instead, reporter genes are often employed to easily... be assayed conveniently. A direct test for your gene of interest may be another method to assess transfection...conjunction with positive selection to ensure your gene gets targeted to a specific location within the ...used selection markers are listed below: Name Gene Conferring Resistance Cell Types* Mode of Action...use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin...Read More: Plasmids 101: Antibiotic Resistance Genes Plasmids 101: Origin of Replication All Plasmids...the genetic traits of the foreign material and will subsequently pass it on to future generations. Stably...

...Imagine you’ve just discovered that your favorite gene was described in a CRISPR screen publication. You...actual data. Now you’re wondering if your favorite gene is included in other CRISPR screens and whether ...researchers to query all public CRISPR results for any gene of interest. In addition, users interested in a ... thresholds used by the authors to determine the gene hits for each screen are also captured as metadata...results are reported as the original quantitative gene-level scores provided by authors rather than sgRNA-level...available resource developed by the Biological General Repository for Interaction Datasets (BioGRID) in...Twitter at @biogrid.     Additional resources on the Addgene blog Read this blog post about genome-wide screening...

...proteins (FPs) to a yeast gene of interest. These fluorescently tagged genes are expressed under native...sequence and a selectable gene product for isolation of successful transformants. Addgene distributes several...be interested in attaching epitope tags to their genes of interest, allowing for easy capture and detection...modifications in S. Pombe, including full and partial gene deletion, overexpression (by promoter substitution...few. Have you used these tools in your own lab? Addgene would love to hear from you, our community, about...

...526) that (1) enables efficient knockout (KO) of genes in immune cells; (2) is not immunogenic, a necessity...for in vivo studies; (3) is compatible with single gene KO and pooled screening approaches; (4) encodes ... optimize conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses...screen, we used the pRDA_526 vector to knock out 30 genes putatively involved in CD8+ T cell responses to ...Markson et al., 2024), developed and deposited at Addgene by Arlene Sharpe’s lab, enables in vivo screens...10.1084/jem.20230699. Additional resources on the Addgene blog X-CHIME: Context-Dependent Germline Knockout...

...REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large gene into smaller...the introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated in an AAV vector (Figure...doi.org/10.1101/2023.12.12.571321.  Revolutionizing Gene Therapy: The REVeRT Dual AAV Vector System By Vaibhav...AAVs, and then splicing their mRNAs, the functional gene product is reconstituted within the target cells...vector-based therapies. REVeRT supports advanced gene expression regulation, enhancing both the mechanisms...trans-splicing dual AAV vectors for (epi)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org...

...available at Addgene CRISPR-Cas9 References, Protocols, and more References: Generation of Gene-Modified ...the CRISPR/Cas9 system to precisely edit specific genes in monkeys. "Our study shows that the CRISPR/Cas9...system enables simultaneous disruption of two target genes in one step without producing off-target mutations...Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos. Niu Y, et al. Cell. 2014...many human diseases are caused by genetic abnormalities, targeted genetic modification in monkeys is invaluable...invaluable for the generation of human disease models." Addgene Outreach Scientist and fellow blogger Caroline...

...variations in gene expression, they found that for approximately 40% of the identified genes, the genes were not...explanation. Next, they hypothesized that differences in gene expression could explain why some ancestry groups...groups were differentially dependent on certain genes. This didn’t prove to be correct, either. However, ...sequences for sgRNAs that target ancestry-associated genes.   What does the reference genome actually reference...cases, this may lead researchers to deprioritize genes that could be good targets for therapeutic development...cells’ germline genetic variations. They then wanted to identify cancer-associated genetic dependencies ...ancestry which will reduce the likelihood of generating genetic dependency data with an ancestry-based bias...

...on vectors where the gene of interest is inserted into the middle of the lacZ gene, thus disrupting 𝛽-...where only cells that have gained or lost a specific gene survive (ex: antibiotic resistance marker). In a... the blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which ...scientists have found that when a portion of the gene is deleted, it creates a non-functional 𝛽-galactosidase...mentioned above. In molecular biology, cloning a gene into your plasmid is often a numbers game. By using...troubleshooting Sanger sequencing results. At Addgene, we now use next-generation sequencing during our QC process....pigment. Find plasmids for blue-white screening at Addgene! Some bacterial strains contain lacZ in their genome...

...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under...design. You can create a new strain with a disrupted gene by transforming wild-type yeast with an integrating...modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check... More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other... is which vector will best suit your needs? At Addgene, we have a vast collection of empty backbones that...vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected cells, you will not need...

...region of the gene they are trying to disrupt or delete, which both inactivates the gene and acts as a...maintain the plasmid. Adding an antibiotic resistance gene to the plasmid solves both problems at once – it...Historically, antibiotics have also been used to disrupt genes at the chromosomal level. Scientists introduce an...typically has two separate resistance cassettes and your gene of interest is cloned into/inactivates or completely...resistance!  What are antibiotics? Antibiotics are generally defined as agents that kill bacteria, or inhibit...lab, their mechanism for killing bacteria, and general working concentrations. For instructions on how...how to prepare antibiotic stocks, see Addgene's Reference Page. How else can antibiotics be used in the...

... gRNAs) that targets ~4,000 E. coli genes with ~15 gRNAs per gene and includes ~10,000 gRNAs targeting...CRISPR interference (CRIPSRi) platform for targeted gene repression in Candida albicans was developed by ... transcription of HSP90 an essential C. albicans gene. Read the paper in mSphere Find the dCas9 and... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR...a new CRISPR tool you’ve recently deposited to Addgene and you’d like it to be included in the next What...more information. Additional resources on the Addgene blog Download the CRISPR 101 eBook Find our CRISPR...posts Read our CRISPR cheat sheet Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids by...

...Energy’s Joint Genome Institute for uncharacterized genes that were proximal to a CRISPR array and the universal...CRISPR-Cas systems containing cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40...-Cas systems. There are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All these...potential of Cas14, the Doudna lab cloned the cas14 gene together with the adjacent CRISPR array and intergenic...presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific of a viral...ID PMID: 30337455. Additional resources on the Addgene Blog Learn more about DETECTR Browse all of the...Download our CRISPR cheat sheet Resources on Addgene.org Find the Cas14 plasmids here Checkout our CRISPR...

... are, in most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet ...infection; however this would not be great for a gene therapy vector! For viral vectors, only the protein... is (thankfully) gutted of many essential viral genes. How it is, exactly, that heaps of viral vector ... particles are made while missing the polymerase gene may seem like a mystery. But it’s simple. The RT...the only component that is not dependent on viral genes. It is derived from the host/producer cell membrane...full “life cycle” of expression of selfish, lazy genes of a non-living viral particle, and how we as dedicated...Kantor, B. (2021). Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State...