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...transcription of other relevant viral genes (including the ones listed previously). E3/E1B suppresses ...applications and aid in ease of use. Our lentiviral guide outlines these components.     Fig. 1 - Viral vector... AAV also requires some back up from adenovirus genes – E4, E2a, and VA – which are supplied on a helper...viral replication. More information on these viral genes is available in the adenovirus section below!   ...

... nucleic acid sequence of the antibody-encoding genes over time as the hybridoma cells divide. These changes...hybridoma cells before culturing or they can clone the genes from the hybridoma into a plasmid to create a recombinant...Figure 2: Changes to the heavy chain or light chain genes within a hybridoma impacts the specificity, avidity...Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B, Aldred SF, Bestagno M, Burrone O, Maynard J, ...1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies. https://www.ncbi.nlm.nih.gov...

...TRIM21 toxicity, Clift et al. created stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression...contain changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not ... within 4 hr of treatment. In some primary cell lines, endogenous TRIM21 is sufficient for TRIM-Away, ...Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid and reversible...that can independently control expression of your genes of interest. These transcription factors consist...

... al. "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities." Cell...et al., 2014; Konermann et al., 2014) individual genes for studies interrogating their functions. The continual...list of effective gRNA sequences targeting your genes of interest. Thanks to the tremendous efforts made...Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell death...

...engage broadly with diverse communities, to raise awareness about where science is and where it is going, ...Harvard Medical School, has a mission of raising awareness and starting conversations about both the benefits...participation in new precision medicine research. A willingness to confront these tough issues is essential for... of individuals from diverse backgrounds and disciplines, and everyone on staff takes on a variety of ...of their families. Having a broad interest and awareness of issues outside of your field of research will...

...lentiviral) generation The first step in delivering genes via lentivirus is deciding what packaging system...VSV-G, a packaging plasmid containing structural genes and a transfer plasmid carrying the transfer gene...) while a single 72h harvest is best for larger genes such as Cas9. See figure 1 for an example of how... can affect transfection efficiency. For some transgenes, transfection optimization simply is not enough...sufficient titer; this is often the case with large genes. This is due to inefficient packaging of large inserts...

...can now precisely control where and when certain genes are expressed to investigate their role in a specific...would behave very differently (distracted) from the ones tested at the beginning. Variability source Standardization...manipulation can also reduce expression levels of certain genes. A popular method uses Cre recombinase on transgenic...vectors are superior packaging vectors for larger genes - they can hold up to 8 kb, while AAV can only carry...Timing: High expression levels of the viral vector trangenes typically occur two weeks after injection. Therefore...

...additional safety benefits: they lack all viral genes, including those that are responsible for integration...significant clinical efficacy against a form of blindness known as Leber’s congenital amurosis (LCA). This...RPE65. It is characterized by childhood onset of blindness and was thought to be untreatable until 2008. ...Furthermore, deeper understanding of disease pathogenesis will enable robust therapeutic outcomes. In ...decline in vision with gene therapy in childhood blindness." New England Journal of Medicine 372.20 (2015...

...into account (Fig 1a). The laboratory of Karmella Haynes at Emory recently contributed a set of plasmids...week. Taking advantage of Golden Gate cloning, the Haynes lab’s Protocols.io protocol describes an elegant...chromatin within a nucleus -> DNA compaction around histones -> Examples of histone-modifying enzymes and reader-effector...reader-effector proteins. b) Golden Gate assembly (backbones available at Addgene) with examples of potential... proteins quickly and accurately.   Priode and Haynes, protocols.io. 2021. dx.doi.org/10.17504/protocols.io.brgcm3sw...

...presenting, it’s no wonder it was attended by over 300 Chinese scientists eager to hear current findings in nucleic...where one can shop for components of traditional Chinese medicine. There were hundreds of stalls selling...Lowe talked about his work on the 500+ human tRNA genes and, until now, we've overlooked regulatory fragments...may be involved in preventing silencing of self-genes. Dr. Mello presented a hypothesis that these pathways...are engineered to replace the drug resistantance genes in the bacteria thus making the bacteria sensitive...

... to summarize these principles it would be “assertiveness.” 7 strategies to become more assertive & resolve...resolve conflicts What Is Assertiveness? An assertive person is able to communicate their ideas confidently...your action item list and set some approximate timelines. Be sure that you keep your commitments, and if... you to follow through on your commitments. Assertiveness is a skill that will help you to resolve both.... An excellent resource to learn more about assertiveness skills is Dale Carnegie’s book “How To Win Friends...

...containing plasmids differing in their resistance genes, as one does not need to prepare separate batches...

...a chance of seeing a phenotype since additional genes that can do the same function are less likely to...Essentially, the X, second, and third chromosomes are the ones you’ll work with. If YFG is somehow on the Y or ...Geneticists need a method for maintaining stable lines of genetic mutants, including in situations where...lose track of your mutation. To maintain stable lines of flies carrying a specific mutation, Drosophila...Drosophila cell line. Knock downs in Drosophila cell lines are extremely effective. Their Snapdragon tool helps...

...contacts that bring apposing membranes together and can wrap the membranes into stacked structures including...every lab in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to...tubules can be grossly distorted into dense stacked membranes termed Organized Smooth Endoplasmic Reticulum ..., M.V., et al., Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol, 1999. 17...

...loci targeted One Many Sample type(s) Cell lines Cell lines, primary cells Table 1: Key differences...multiple targets because different CAPTURE cell lines will have varying levels of dCas9 and gRNA expression...components eliminates the need to create stable cell lines and allows CAPTURE 2.0 to be used with primary cells...like to interact with, and how many promoters or genes each enhancer is interacting with. Super enhancers...

...SB_loop, SB_convex. These scaffolds contain serines and threonines at positions that can be randomized for...epitopes that are hard for antibodies to get to, like ones that are hidden in the molecular crevices proteins...Developing antibodies against proteins embedded in membranes is tricky because they have fewer hydrophilic ...diverse pool of sybodies that hypothetically contain ones that bind strongly to your target protein. The Seeger...

... AsLOV2. After several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet variants...significant gains! After a few more rounds of mutagenesis, they eventually found a version of AsLOV2 with...changes under magnetic field. Such changes could be harnessed or enhanced for future MagLOV-based tools, just... Ingaramo, M. (2024). Magnetic control of the brightness of fluorescent proteins. Zenodo. https://doi....., & Kusamoto, T. (2023) Single-Molecule Magnetoluminescence from a Spatially Confined Persistent Diradical...

...Origins of protein tags For about as long as the disciplines have existed, cell and molecular biologists have...developing generic strategies for detecting cloned genes (Munro and Pelham, 1984). The strategy they landed...1985). Myc was one of the earliest identified oncogenes and was a target of intense research. Evan, et...explore Myc’s role in cell proliferation and tumorigenesis.  Using two different synthetic peptides corresponding...tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila...

...(1x cysteine) pTP955 (3x cysteines) Mouse IgG1 TP886 pTP943 (3x cysteines) Mouse IgG1 TP1107 pTP1112...1x cysteine) TP1122 (3x cysteines) Mouse IgG2a TP1129 pTP1005 (3x cysteines) Mouse Kappa TP1170 ... pTP1174 (1x cysteine) pTP1172 (3x cysteines) *Rabbits have only one IgG subclass.   Express the ...

...” is the title of the chapter that examines the inherent brokenness of scientific publishing and research...tackles much broader issues in this brief volume. He examines, amongst other topics: racism and sexism in science...the U.S. environment, and a decline in the competitiveness of U.S. science and technology.” This last ...in under 200 pages is an impressive task to set oneself. As a scientist who enjoys learning a little bit...

...Plasmids need antibiotic resistance genes so that you can specifically isolate bacteria containing your...action of the most common antibiotic resistance genes and which applications are best (or worst) for each...plasmid of interest, preventing this issue. The genes and their antibiotics   Ampicillin Ampicillin, often...antibiotic due to stable integration of resistance genes in their genomes. If you need a plasmid that can... Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage...

...breast and ovarian lines commonly used in lab. It is recommended you check your cell lines for HR-related ...covered in this blog, with tips and tricks for harnessing the homology-directed repair pathway, designing...surprise then that these edits take more planning and finesse then their knock-out counterparts. To kick off ...experiment today! Get ready to identify knock-in clones by reading up on validating genome edits to find...

...biological disciplines and scientists employ GFP for a wide number of functions, including: tagging genes for...Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed...began engineering new versions of GFP through mutagenesis in order to improve its physical and biochemical...Click here to view Addgene’s collection of empty backbones for constructing fluorescent fusions. Transcription...

... help you verify the ends of the insert. Longer genes may require internal primers that anneal to a specific...

...digests needed in molecular biology, and thaw cell lines. There are many different types of water baths that...

... propagation of themselves and their associated genes. These systems better ensure that plasmids are passed...death. The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed... (hok), a long-living toxin that damages cell membranes; Suppression of killing (sok), a short-lived RNA...proof of concept, they tested a lux-mediated bioluminescence reporter in vivo in the absence of selective... Ogura, Teru, and Sota Hiraga. "Mini-F plasmid genes that couple host cell division to plasmid proliferation...

...AAV enhancer plasmids with high specificity and brightness, now available at Addgene!   {% module_block ... those specific cell types to express exogenous genes — a feature the AIBS has capitalized on. ...containing enhancers with the highest specificity and brightness. You will find enhancers to target many different.... A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types. Cell...., 288–295. https://doi.org/10.1038/nrg3458 Ray-Jones, H., & Spivakov, M. (2021). Transcriptional enhancers...

...To deliver genes using lentiviral vectors, you need an envelope protein on the virus’s surface and a ...stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal... were less stable than viruses produced in cell lines that do not express these sugars. (Takeuchi et al...study the spike protein’s role in infecting cell lines without using intact SARS-CoV-2 virus. Scientists...find tables and information on different viral membranes, tropism, and advantages and disadvantages: Table...

... similar brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP...Master. Note: BRET= Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer...that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry...

... are essential for processes such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction...reactive and forms a link with nearby nucleic acids (McInnes et al., 1990). Photobiotinylation is sometimes ...18988027. PubMed Central PMCID: PMC3223083. 6. McInnes, J. L., Forster, A. C., Skingle, D. C., & Symons...Biotinylation ThermoFisher Scientific: Biotinylation Guidelines  ...

...Repair is Used to Edit Genes Read How Non-Homologous End Joining is Used to Delete Genes Additional Resources...exons), but much of the sequence appears to be nonessential. Within the mutational hotspot for dystrophin... cells expressed functional dystrophin. Editing genes in vivo is of course much more difficult than in...these different mutations. For each therapy, effectiveness and off-target risk must be evaluated separately...

...Isolation and Concentration (MagIC)-cryo-EM that combines enrichment and imaging of proteins captured on...with a GFP nanobody (Figure 1). This enrichment streamlines the purification and minimizes sample loss while...loss. Ci Ji Lim’s lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins that... to partially correct mouse models of genetic blindness (An et al., 2024) — the first demonstrated use...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...plasmid yield and/or DNA quality. Table 1 below outlines a few of the more common genetic changes found...encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type unless indicated otherwise...lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced, expression...transformation efficiency deoR constitutive expression of genes for deoxyribose synthesis Allows uptake of large...)]  Note: Inactivating mutations in specific genes are signified with a minus sign (-) as is typically...

... and may provide counter-selection for negative clones as in the example shown here. The manufacturer ...

...Finally, as Pamela Hines of AAAS points out on the panel, irreproducibility undermines public trust in science...Last Wednesday we worked with the Harvard GSAS Science Policy Group to organize a Minisymposium on Reproducibility...) - Panel Alex Tucker (Ginkgo Bioworks) Pamela Hines (Senior Editor at Science) Edward J. Hall (Professor...journalists and scientific experts discussed by Pamela Hines Minisymposium on Reproducibility Booklet - Including...

...involves making a large sgRNA library to thousands of genes and targeting and sequencing millions of cells. ...cell line, with the goal of isolating single cell clones to use for multiple downstream experiments. You...certain Cas enzymes are less compatible with the bulkiness that accompanies mispairing of bases. To reduce... characterization of, isolating 2 or 3 distinct clones to perform key experiments on is a good way to ... distinct off-target sites, eliminating its effectiveness as a control. Since off-targets typically confound...

...al., 2009), and at high copy numbers (Williams, Carnes, and Hodgson, 2009). More research is ongoing to...1566524020666200427110452. PMID: 32338220. Crozat E, Fournes F, Cornet F, Hallet B, Rousseau P. Resolution of...Levene SD. Analysis of DNA topoisomers, knots, and catenanes by agarose gel electrophoresis. Methods Mol Biol...0092-8674(84)90060-6. PMID: 6323019. Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design... JA, Luke J, Langtry S, Anderson S, Hodgson CP, Carnes AE. Generic plasmid DNA production platform incorporating...

...many of which are used to deliver Cas9 and other genes to mammalian cells. For this reason, the top requested...means researchers who use VSV-G can deliver their genes of interest to a variety of cells and study their...Skimming the list, one gets a sense of the broad usefulness of pMD2.G and VSV-G. This plasmid has been used... of the secretome, ubiquitin ligase, cellular senescence, autophagy, CRISPR, and so much more (find the...

...edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post...protein-coding capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both...Veres et al., 2014). When working with single-cell clones, the authors note that “clonal heterogeneity may...obstacle to the generation of truly isogenic cell lines than nuclease-mediated off-target effects.” Further... CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem ...

...societal implications even before the discovery of genes themselves.  How has scientific progress in genetic...and vice versa? We draw a parallel between the timelines of the scientific advancements and the noteworthy...to stars in space ships and the rise of killer machines that exterminate entire humanity were the two ...that results in gene knockouts.   Star Trek: Into Darkness (2013) which is the remake of the space-melodrama...Hugo De Vries: from the theory of intracellular pangenesis to the rediscovery of Mendel. Comptes Rendus ...