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... discriminate between positive and negative clones. Clones with insertion will show only linearized plasmid...: Can you summarize HR template vector design guidelines? A7: A few notes below are considerations for...plasmid of ~8.5 kb (only AgeI will be able to cut). Clones without insertion will show a ~1 kb and ~7.5 kb...) on each colony in order to screen for positive ones.  -- OR -- If your HR template has a selection... primers that may work better than the published ones, so if/when you use HercII or Kapa Hifi the reaction...During Gene Targeting with Zinc-Finger Nucleases. G3 Genes Genomes Genetics, 3(4), 657–664. https://doi.org...generation of mice carrying mutations in multiple genes by CRISPR/CAS-Mediated Genome Engineering. Cell,...

...lifecycle. Halloween genes The Halloween genes are a set of cytochrome P450 genes discovered in Drosophila...Mutations in these genes are lethal, associated with defective exoskeleton formations. These genes were appropriately...1983): 813-816. Gilbert, Lawrence I. "Halloween genes encode P450 enzymes that mediate steroid hormone...

...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 ...Streptococcus thermophilus (the popular Streptococcus pyogenes system does not work well in Caulobacter). To ...system can also be used to downregulate multiple genes at once. Read the paper in mBio Find the CRISPRi...transgene design and delivery. They generated hPSC lines with AAVS1-integrated, inducible, and fluorescent...which increases activation of methylation-silenced genes. Read the paper in Nature Communications Find Casilio-ME...

...lentiviral backbones designed right here at Addgene. Here's what makes these new backbones special: Strong...deactivation rates Addgene's own empty lentiviral backbones New recombinant antibodies for Glypicans DNA editing...deactivation). Both have significantly improved brightness, kinetics, and sensitivity. iGluSnFR4f is well-suited... Build your way: Explore Addgene's new empty backbones By Meghan Rego You asked, we listened! One of ...favorite features and limitations of existing backbones, we're excited to share our very own set of empty...labeling By Alyssa Shepard To fully understand how genes are regulated, it's important to know what proteins...

... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...genome editing system derived from Streptococcus pyogenes. An alternative Cas9, from Streptococcus aureus...effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage of being smaller (Zhang ...C57BL/6, for which we have germline competent ESC lines. Second, the process is much quicker. It takes 3...Wang is a professor at the Institute of Zoology, Chinese Academy of Sciences, and he is interested in using...Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell ...

...challenge when researchers are communicating across disciplines or doing different tasks. But these challenges...can be exaggerated when communicating across time zones, digitally, and asynchronously. The best way to ...collaborating with remotely. Depending on the willingness of the team to adopt various tools, this may ...collaboration includes scientists working in different time zones, it’s important that everyone can access data and... collaboration. Some things to figure out are timelines for experiments and manuscripts, who is responsible...your recurring meetings and reaching out if any milestones are missed. Spend time in person Presenting...

...thanks to our guest writer, Pamela J. Hines, PhD! Pamela J. Hines is a Senior Editor at Science magazine... post was contributed by guest writer Pamela J. Hines PhD, Senior Editor at Science Magazine. Although...replication and gene transcription during vertebrate embryogenesis. My interests, though, have proven unruly. Instead...journalism? Take a look at our news editors. A fondness for visualization of science? Look at our graphics...French horn, and rhubarb. Follow her on Twitter @Pam_Hines and LinkedIn. Additional Resources on the Addgene...

...graduate student, you are in a system that, quite honestly, does not value your potential contribution to... science, you are likely lacking the necessary business background and/or connections to even begin this...pitch what you hope to build.  Then ask for their honest opinions -- this part might hurt, but if you pitch...made, we considered the feedback and adjusted our business model accordingly until we had a model that was...This will allow you to discuss each project and business decision before making it, to have an internal...faculty, venture capital and angel investors, and business development heads at major companies.  The majority...

...signaling is likely to have). Anterograde tracing outlines neurons from their cell bodies to the terminals...terminals of their axons; while retrograde tracing outlines neurons in the opposite direction, from the terminals... dynein, while anterograde transport relies on kinesin. Different rates of speed for various forms of ...predominately anterograde tracing, biotinylated dextran amines (Veenman et al., 1992) or fluorescent molecules... achieved by using optogenetically controlled transgenes, in combination with conditional transgenic mice... M., and Schnell, M. (2008). Concepts in the pathogenesis of rabies. Future Virol 3(5), 481–490. PubMed...Ho, A., Nicholas, E., Hirokawa, K.E., Bohn, P., Joines, K.M., Peng, H., Hawrylycz, M.J., Phillips, J.W...

...MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural precursor protein...tropisms.  Packaging using helper-free packaging cell lines This method utilizes a packaging cell line that ...and/or Env, eliminating the need to deliver these genes in trans by specific plasmids. This method, therefore...standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are different viruses...replication incompetent? Yes, as with lentiviruses, the genes required for γ-retroviral packaging and transduction...element; sequence that stimulates the expression of transgenes via increased nuclear export. LTR Subcomponents...

...nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including 1... knock it out, etc. It’s how we understand what genes do! Viruses have several options to allow you to...engineered to introduce frameshift mutations in genes of interest, turning expression off permanently....2007.   Clinical applications Viruses in vaccines helped pave the way for their use in other clinical...The use of viral vectors in vaccine development. Vaccines. 7, 75 (2022). DOI: https://doi.org/10.1038/s41541...

...virus will target and fuse with. In nature, this determines which organisms and cells they infect. In the...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...

...more commonly used backbones and specific primers you can use to confirm your clones. You can also find...screen will help you more easily identify successful clones so you have to weed through fewer colonies after...antibiotic resistance. But what about identifying clones that contain your desired insert? This is where...how you can use restriction digest to screen your clones, but there are many other ways to choose restriction...PCR.   Sanger sequencing Sanger sequencing determines the precise order of nucleotides within a DNA...

...all known genes. This is a perfect example of an unbiased, reverse screen – all known genes are assayed... a biological process. Players can be proteins, genes, enzymes, drugs, or anything else that plays a role...where there is removal of known factors (such as a genes) and the loss of those factors is assessed. The ...similar to CRISPR, but instead of knocking out the genes of interest (or the whole genome), they knock down...especially useful if you are interested in studying genes whose knockout would be lethal to cells and thus...

...on cell lines susceptible to or dependent on certain media components, and are rescued by genes supplied...colonies (used to complement auxotrophic cell lines), or genes which rescue a lethal phenotype. Resources... transformed plasmid. Such plasmids may contain genes that allow for the use of a particular substrate...list of Bacterial Expression Systems Find Empty Backbones for Your Experiments    ...

...number of genes involved in cardiovascular and metabolic diseases. We are knocking out genes that have...mutant versions of 15 different disease-associated genes. Addgene spoke to Musunuru about how the new kit...kit is quite useful in generating the mutant cell lines that one needs to do disease modeling in stem cells...several mutations in the same gene or in multiple genes, all of which seem to contribute to a particular...iPS) cells from patients and try to compare those lines, they may all have the mutations but they will also...

...producing landslide after landslide, only adding more stones to the field. Fortunately, our bodies have been... (Th) cells. These cells produce inflammatory cytokines - interleukin (IL)-12 and interferon gamma (IFNγ... promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates the...cancer. Surgical removal causes increases in tumor cytokines like TGFβ and IL-6. However, when strengthened...response. Antibodies targeting the tumor-augmenting cytokines produced in the tumor microenvironment TGFβ, IL...necroptotic tumor cells have also been tested as vaccines and therapeutics. Their uptake by DCs is increased...marker blockade and suppression of tumor-aiding cytokines, for example, will more broadly activate the immune...

...either through a chromogenic or chemiluminescent reaction. Chemiluminescence, which is light produced by ...the primary amine groups from lysines, sulfhydryl groups from cysteines, or carbohydrates from glycosylation... found throughout the antibody. 2) Endogenous cysteines, such as those that make up the disulfide bonds...antibody (Mueller, et al., 1988). If some of those lysines are in the antigen binding region or even just ...

...experiments. Test dTAG molecule toxicity in new cell lines Treat parental cells (or cells expressing a control...FKBP12F36V-fusion chimera of interest. In the cell lines we have evaluated to date, we observed little to...FKBP12F36V fusion chimera degradation. In the cell lines we have evaluated to date, we have not observed ...was employed to evaluate degradation of ENL. Cell lines were engineered to express ENL-FKBP12F36V and endogenous... employed to evaluate degradation of MELK. Cell lines were engineered to express FKBP12F36V-MELK and endogenous...was employed to evaluate degradation of YY1. Cell lines were engineered to express YY1-FKBP12F36V using ...

...are tumor microenvironments made of?  A tumor’s genesis is divided into 3 phases: initiation, progression...active regions, microenvironments shape a tumor’s evasiveness, evolution, and response to treatment. Some microenvironment...transport miRNA from drug-resistant cells to sensitive ones. Though this process is not fully understood, a ...formation by inhibiting cell growth, invasion, and angiogenesis (Allinen et al., 2004). In fact, Sanford H. ...influence tumor microenvironments: immunology and angiogenesis (Witz et al., 2009). Then, scientists began ...Fernando Schmitt. "Microenvironment in breast tumorigenesis: Friend or foe?." Histology and histopathology...