...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...

...frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock ...

...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 ...

...Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified...Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of most CRISPR...gene that made it less effective in certain cell lines. This point mutation has been corrected in V2.0....gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain GFP-tagged...

...overview of the instrumentation A flow cytometer combines three systems to analyze single cells from a mixture...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...targets, measured by fixing and permeabilizing membranes For intracellular protein targets, users must ...propidium iodide, can only enter cells with disrupted membranes as is the case with dead or dying cells. DNA binding...the cells, amine reactive dyes interact with free amines in the cytoplasm causing dead cells to fluoresce...

...visualize the protein via fluorescence or chemiluminescence, precipitate the protein out of solution, ...region   The constant region of the heavy chain determines the antibody’s isotype - that is, one of five...which come from naturally-occurring immunoglobulin genes, recombinant antibodies are plasmid-based. Their...antibody allows the protein to be detected by chemiluminescence or fluorescence. Western blots are frequently...Steven GE, Lorna J. (2004) ANTIGENS, IMMUNOGENS, VACCINES, AND IMMUNIZATION. In: Immunology Guidebook. Elsevier...

...screens using CRISPR technology, gRNAs to knockout genes – are essential screening components, the model ...Sometimes, you might have a few positive control genes on which to test the validity of your biological... a new assay or both, and you have no idea what genes are involved. Or you don’t know how well your assay...definition, the kinome is a well-annotated collection of genes that can provide a useful starting point for developing...

...Directed Mutagenesis by PCR Google Forums Round Up: First Impressions of NgAgo CRISPR Between the Genes: How...optimize and troubleshoot the system. Site Directed Mutagenesis by PCR & Single Base Editing with CRISPR For ...technology may make some uses of Site Directed Mutagenesis by PCR obsolete! Honorable mentions Of course...

...miss out on beneficial opportunities or to find oneself unsure of the next steps. Harvard PI Angela DePace...lab culture. The aim of these meetings is to give honest, constructive feedback, set tangible research and...personal development through thoughtful planning and honest feedback DePace’s yearly planning meetings revolve...person organize his or her thoughts to facilitate honest conversation. Goals and Planning Worksheet: ...

... more often than other disciplines. The majority of tweets for most disciplines contain scientific domains...#8. Figure 1: Highly Represented Scientific Disciplines on Twitter. Twitter users were identified as ...Follower networks of subgroups of scientific disciplines: social (left), life (center), and physical (...

...based on a protein isolated from Streptococcus pyogenes that locks itself together, to produce irreversible...another important step forward by dissecting that S. pyogenes protein into three parts. Their efforts yielded... from the “S” in Streptococcus and the “py” in pyogenes), capable of locking two peptide tags together... chain.  Addgene: What about the Streptococcus pyogenes protein domain made it ideal for this purpose?...

...into the following categories: Over 300 empty backbones with 100+ distinct fluorophores to choose from...constructs for biosensing. Over 300 plasmid backbones spanning a rainbow of fluorescent proteins “It...It’s a great collection with over 300 backbones alone,” said Addgene’s Lianna Swanson, who has been working...collection with such variety.” In most cases, backbones are available to attach the gene of interest to...

...currently houses over 890 CRISPR screens in 629 cell lines, each manually curated and annotated from more than...and power of CRISPR-based library screens in cell lines from humans and other species. By accurately compiling...browse through screens performed in relevant cell lines or conditions. This increased ease of access not...screens performed in 26 different breast cancer cell lines. Screenshot updated 2024. Each screen curated...

...all know that the Fc domain provides structure, determines isotypes, and provides a place for secondary ...cannot survive when holes are punched in their membranes, the pore'd pathogens quickly lyse and die.  Antibody...induced “stabbing” and “poisoning,” but let’s be honest, the elimination style the immune system is most... that!    References and resources References Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory...

...two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type AAV, these two genes are...which contains the AAV structural and packaging genes, the adenoviral helper plasmid which contains the...Next let’s take a closer look at the rep and cap genes. Figure 1. The RepCap plasmid and its products...

...Giving honest and helpful feedback gets easier with practice so you must build it into your routines. This...their work.  Check out Joanne's Reddit AMA   Guidelines for giving feedback Use formal and informal ...differently? Beware of indirect language You must be honest about performance problems. While giving corrective...

... Encodes insert(s) A plasmid with one or more genes, protein coding sequences, or other elements inserted...designed for cloning a gene/insert into it. Empty backbones often contain epitope tags or fusion proteins ...Insert *Note: This page will not appear for empty backbones Please enter the name of any gene contained within...Addgene ID in the "Backbone Manufacturer" box for backbones requested through Addgene. For example, you could...Addgene qualifies as BL1 or BL2 as defined by NIH guidelines. I hereby certify that I have disclosed all known...

...capable of robust expression of high G+C content genes, as validated through monitoring the expression ...adapted for RNAi experiments for mammalian cell lines. The Turner Lab Human messenger-RBP sgRNA Library...MYC-ChIP-seq data from several MYC-dependent cancer cell lines. It can be applied for identification of essential...pooled library from Junjie Chen's lab targets human genes known or suspected to be involved in DNA damage ...

...issues by creating immortal antibody-producing cell lines (hybridomas) that secrete antibodies in culture ...production. However, even clonal hybridoma cell lines can produce more than one mAb (Bradbury et al., ...hybridomas can lose gene expression of the mAb-encoding genes, die, or fail to revive after cryopreservation (...Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B, Aldred SF, Bestagno M, Burrone O, Maynard J, ...

...Kamens several years ago through the Healthcare Businesswomen’s Association and it was through this connection... systematically test the function of all 20,000 genes in disease models and then focus drug discovery ...early age, I was fascinated by the impact that medicines can have on human health. As a teenager in high...the field of gene therapy.” He’s also worked on vaccines throughout that time. Now Vandenberghe is an Associate...

...raw plasmid sequence and display frequently used genes and control elements. Development of this tool required...feature sequences due to genetic drift and the use of genes from different microbial strains. It proved to be...sequence feature that may be used to make fusion genes, detection needs to occur even if one or two codons... our database, or if you see an opportunity to harness common feature detection in a new way, please contact...

... Addgene to gene-edit mutations into XP genes and other genes involved in photosensitivity in melanocytes...hemiplegia) that are often painful and accompanied by migraines. Those attacks can be brought on by a number of...plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models carrying different SOX10 mutations...

... cell type of interest better than immortalized lines derived from the same cell type; however, their ...interest into primary cells, so most primary cell lines require viral infection. A new paper from Niels ...alter protein function or localization. Thus, the usefulness of CPPs must be evaluated on a case-by-case basis...biallelic mutations. Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR...

... delete/overexpress endogenous genes or to introduce heterologous genes, but the trend of research has...mimic natural transcriptional programs on multiple genes. Modifying the CRISPRi system for multidirectional...programs to differentially alter the levels of five genes that encode biosynthetic enzymes for violacein, ...

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

...keeping the system to minimal size. To do so, they harnessed the tRNA processing mechanism, which is conserved...lentivirus, demonstrating simultaneous editing of genes against multiple genetic diseases, such as muscular...for correcting mutations or installing functional genes to study and potentially treat polygenic diseases... that would perturb the unprecedented number of genes with a single viral vector.   Fig. 1: Schematic...

...medical conditions had been swapped with made up illnesses. They were then asked to evaluate the quality ...methodology makes it easier to spot even minor weakness or limitations in a piece of research (Drummond... mask designs could work to slow the spread of illness. I later helped run workshops where I showed people... jargon that even scientists in neighbouring disciplines might struggle to understand. Why should we trust...

...screens, enabling genome-wide interrogation of the genes required for cellular processes. For a successful...that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation, ...-LPs and accelerate the discovery of additional genes and small-molecule probes to aid in the understanding...to different lipid compositions of C. elegans membranes, which might beneficially influence the rhodopsin...

...assignment to discuss goals, expected outcomes, timelines and deadlines. Agree on a way to review project progress...identify constraints and boundaries. Where are the lines of authority, responsibility and accountability?...try "Why Aren't You Delegating" in the Harvard Business Review. Another excellent blog on delegation tips...

...and funders in the Transparency and Openness Promotion Guidelines; preregistration to increase clarity...variable actually makes a big difference for the robustness of the findings.   How do we make research more...code, and other materials. The use of reporting guidelines can also help remind us to include all of the...

...learn new tasks and beat humans at the ancient Chinese game Go). In addition, there was an entire story-telling...neuroscientists are using techniques from across different disciplines to better understand neurobiology and human cognition... provide a highly versatile means of delivering genes to many different cell types with minimal immunological...effects. AAVs are used in experiments targeting genes to specific cell types in the brain, mapping neuronal...

...cloning uses short sticky ends to insert genes into plasmid backbones, base pairing between these ~4 base ... different types of PCR-generated inserts. By harnessing the power of DNA repair, you can assemble multiple...2024.  References and Resources Further reading Harnessing homologous recombination in vitro to generate...

...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...PAM sequence that the Cas nuclease recognizes determines site accessibility. Cas9s with a broader PAM ...Joung and Jennifer Doudna Labs used targeted mutagenesis to generate hyper accurate Cas9 (HypaCas9). This... sites. Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created pX-evoCas9...

...observe a phenotype, the modification of multiple genes is often necessary. Guide RNAs (gRNAs) are commonly... al constructed polycistronic glycine tRNA-gRNA genes (PTGs) in the forms tRNA-gRNA and tRNA-gRNA-tRNA...constructed PTGs containing 8 gRNAs targeting 4 MAPK genes, again using rice protoplasts. Each pair of gRNAs...knockout, or multiple gRNAs may be used to target genes with overlapping activity. Multiple gRNAs can also...

... 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...

...systems including the popular S. pyogenes Cas9 (Rauch et al., 2017, Hynes et al., 2017). However, Acr inhibition...What’s great about CASANOVA? It works with the S. pyogenes Cas9, dCas9-effector fusions, and xCas9, and requires...compatible approach to simultaneously clone two genes-of-interest (GOI) into two independent expression...