...bacteria, transposons can move antibiotic resistance genes between plasmids or from plasmids to the bacteria... and mechanism of replication. They contain two genes: gag and pol. The pol polyprotein encodes the reverse...transposon in vertebrates. It was discovered in Japanese medaka fish because its insertion into the fish... how they can be used in the lab. Transposon mutagenesis screens Transposons, by their nature, are mutagenic... elements, which makes them a great tool for mutagenesis screens that detect loss-of-function or gain-... these screens, the transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons...to consider when selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best...

...perturbations that may play significant roles in cancer pathogenesis. Their work culminated in the first functional...help cancer researchers studying an assortment of genes. Using patient data to identify mutations of unknown...mutations with strong phenotypic effects can mask weaker ones. Thus, the need for a faster and higher-throughput... team identified 1,049 perturbations in several genes spanning 33 cancer types. The types of mutations...lentiviral constructs using the High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB) technique (.... Plasmids carrying the corresponding wild-type genes were constructed as well. They called this library.../F3 and MCF10A are growth factor dependent cell lines and any mutations that that confer proliferation...

...Syndrome (a disease that results in combined deafness and blindness) is caused by defects in the gene MYO7a...Jean Bennett started working on ways to deliver genes to correct genetic conditions over 25 years ago ...to the identification of several disease-causing genes, including the RPE65 gene responsible for an autosomal...autosomal recessive, severe, childhood-onset form of blindness name Leber’s Congenital Amaurosis (LCA). Shortly...treatment, famously visited Congress to help increase awareness about the potential of gene therapy!). It would... packaging capacity is well suited for larger transgenes. Unfortunately it has limited tropism in the ...used in clinical trials, a variety of engineered ones have shown great promise in preclinical studies ...

.... Maintaining openness and looseness of spirit The second crucial element in harnessing the power of serendipity...beyond its confines. The beauty of serendipity often emerges at the intersections of disciplines. Cast your... serendipity is maintaining an openness and looseness of spirit. Amidst the rigors of PhD studies and ...routine. It encourages you to think beyond the confines of a predefined path, opening doors to novel ideas...expected. By widening your search and maintaining an openness to unexpected possibilities, you not only invite...

... the paucity of Cre-dependent mouse lines for overexpressing genes within discrete cell populations makes...libraries. However, using CRISPRa to target individual genes can be relatively laborious because multiple plasmids...several sgRNAs must be screened to identify effective ones. Simplified in vivo delivery using SPH mice CRISPRa...neurons over other promoters, including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus...resulted in hundreds of differentially expressed genes between Bdnf I-overexpressing neurons and Bdnf IV-overexpressing...IV-overexpressing neurons. Among the upregulated genes common between the Bdnf I and Bdnf IV overexpression...overexpression variants were other immediate-early genes associated with neuronal activity, such as Fos, Egr3,...

... for a molecular biologist who wants to express genes from one organism in another? Let’s take a look ...correlated with codon bias across all endogenous genes in E. coli and S. cerevisiae (Tuller et. al., 2010...usage frequency in a reference of highly expressed genes (Sharp and Li, 1987). In 2000 the Ikemura lab created...an electronic dataset for codon-usage of 257,468 genes across 8,792 organisms (Nakamura et al., 2000).  ...2017. Most commonly, scientists codon optimize genes from eukaryotic organisms for expression in prokaryotic...optimizing a human gene for expression in E. coli. Genes are also optimized for expression in mammalian cells... lab optimized the C. elegans GluCl ion channel genes for expression in mammalian cells. This was the ...

...Addgene also has ready-made entry clones available for many popular genes, including Hs.KRAS4a. Use our website...selection Used to clone attB-flanked genes of interest to generate entry clones Entry vector attL sites for...competent E. coli cells and select the positive clones. The entry clone and destination vector carry different... recombination step. Addgene’s ready-made entry clones can be used with a large variety of plasmids. Speed...for recombination Used to generate entry clones by TOPO cloning or by Restriction Cloning Destination...the gene of interest in the appropriate system Recombines with the entry clone to generate an expression...plasmids with Addgene. These can be used to express genes in a variety of model organisms. Use the links below...

...CRISPR-Cas can harness the power of MoClo to expand both targetability and effectiveness. Perhaps the most...and Plant Parts Kit III (Gantner et al., 2018; Chamness et al., 2023). The Plant Parts II kit has a focus...for both monocots and dicots (Hahn et al., 2020; Chamness et al., 2023; Stuttmann et al., 2021; Grützner...CRISPR components expressed from different plasmid backbones. They noted that backbone differences such as ...Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. 2017. Crossref, https://doi.org...Crossref, https://doi.org/10.1021/sb4001504. Gantner, Johannes, et al. “Peripheral Infrastructure Vectors and... https://doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit...

...: rare. “Nature has many genes where there are not rare codons and many genes where there are instances...trend of sourcing genes from distantly related organisms. In 2006, less than 1% of the genes deposited at ...synthetic or natural with 97.7% accuracy. Classifying genes as natural or synthetic But how can you tell if ...in a test set of 173 known synthetic and natural genes.  In designing and optimizing their algorithm, ...synthetic. But nine years later, over 20% of the genes deposited that year were synthetic. They also found... Proteobacteria - the most frequent transfer of genes is from Proteobacteria to mammalian expression vectors...Thompson. "Gene synthesis allows biologists to source genes from farther away in the tree of life." Nature communications...

...experiment. Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent Logic in... cell. These so-called recombinase-based state machines could potentially be used to report upon and/or...post to learn more about recombinase-based state machines and how they function. Adapting Toehold Switches...replication and transcription as well as reporter genes. To learn how you can use minigenomes in your research... describes R-bodies, “force generating protein machines that can extend and retract over many microns ...also showed that R-bodies can rupture E. coli membranes. Read the full post for potential applications...Adeno-associated viruses (AAVs) are commonly used to deliver a genes of interest to a variety of cell types in-vivo. ...

...unstructured peptide and therefore consisted of glycines, serines and other small, non-aliphatic amino acids...fluorescent proteins to oligomerize is known as “stickiness.” Since the fluorescent tag should operate as... per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration...oligomerization in cells. In addition to measuring stickiness, several researchers have also collected data...fluorescent protein characteristics such as the brightness, photostability, maturation time, and acid sensitivity...characteristics are important, but have less priority than stickiness. In my experience, fluorescent proteins that ...mediated HDR) enables the tagging of endogenous genes close to native expression levels. However, it is...

...consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product of ...Molecular brightness vs. iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa ...coefficient) (Shcherbakova et al., 2015). The effective brightness reflects the concentration of BV-containing molecules... photoreceptors have higher apparent cellular brightness than previously reported widely used dimeric ...not yield to (m)iRFPs in cellular and molecular brightness (Table 1).  NIR reporters and biosensors NIR ...-the-art NIR FPs, which combine high cellular brightness and monomeric state, are highlighted in blue....Oliinyk, O.S. & Verkhusha, V.V. How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian...

...the secretory pathway. Those can include cytokines and chemokines; enzymes like kinases, phosphatases, and...interchangeably to refer to any technique that combines a marker with a signal. For clarity, Addgene uses...of such targets include transcription factors, histones, and DNA repair enzymes. Note that when using ...label cytosolic proteins alongside the intranuclear ones. You do not need to use a separate fix/perm cytosolic...non-antibody-based stains that bind DNA or free amines of proteins. Depending on the application, they...membrane-impermeant, and the cells of interest can be the ones that are positive or negative for the dye. The main... can only enter cells with compromised plasma membranes — i.e., dead cells — while live cells are protected...

...between MARCH5 and WSB2 genes using Cas12a’s ability to knock out multiple genes in parallel. Lastly, this...individually (~20,000 lanes/50 mLs) or through two polycistronic expression vectors (3570 lanes/50 mLs) coexpressing...frankenbodies to amplify the signal.  Liu et al. Genes Cells. 2021. https://doi.org/10.1111/gtc.12893, ...screening methods have primarily used Cas9 to knock out genes, researchers at the Genetic Pertubation Platform...library. Overall, Cas12a’s ability to target multiple genes within a single cassette provides increased simplicity...individually expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins...gene activation for both reporter and endogenous genes. It is highly specific in mammalian cells without...

...Research the brightness of your fluorophores before buying and try to inversely pair brightness with abundance...sensitive moment.   Fig 1: Blugene fading into darkness because they accidentally left their antibodies...Type Westerns and ELISAs mostly use HRP, a chemiluminescent conjugate that can be activated to emit light...we'll keep it simple. If you follow the two basic guidelines below, you’ll likely be able to create a small...small, usable panel.  First, fluorophores vary in brightness. You don’t want a super-bright fluorophore, like...good match for your experiments, and avoid the awkwardness of getting the right antibody with the wrong ... Boston University Flow Core. Fluorochromes Brightness Chart. Boston University Flow Core. Retrieved...

...lab that expresses the HIV gag, pol, rev, and tet genes. It can be used with 2nd or 3rd generation lentiviral...the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA scaffold and cloning sites...Addgene!) pX330-U6-Chimeric_BB-CBh-hSpCas9 - The S. pyogenes Cas9 (SpCas9) has been widely popular in genome...genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using the BbsI restriction...cassette that makes it less efficient in some cell lines. pSpCas9(BB)-2A-Puro (PX459) V2.0 was made available...from the Trono lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope expressing...lentiGuide-Puro - This plasmid expresses the S. pyogenes CRISPR chimeric RNA element with customizable ...

...achieve success in this business. A good presentation can help win a business plan competition and advance...LabSpaces Blog Find a Buisness Plan Competition Good Relations - PR for the Biotech Business - Posted on Nature.com... Dr. Mike Koeris, co-founder and current VP of Business Development & Operations of Sample6, for advice...2009 and tested in the “market” by competing in business plan competitions. After much learning, the focus... investors to the next stage of diligence." In business competitions everything is conceptual, and the...focusing on concept development and participating in business competitions. When the team was unable to secure...became first COO, then moved on to become VP of Business Development & Operations. Starting a biotech...

... and release IL-36 cytokines. Keratinocytes are the main source of IL-36 cytokines in the skin making ...surrounding IL-36 biology. “Little is known how IL-36 cytokines actually get out of keratinocytes and induce their...researcher at University Hospital Zurich. IL-36 cytokines are a potent inducer of an autoinflammatory loop...Congratulations Mark Mellett! Siddharth Prakash: X chromosome genes involved in Turner syndrome Turner syndrome (TS)...can lead to cardiac developmental defects. Eight genes from the X chromosome are expressed at significantly...from Addgene, the lab will express these candidate genes in TS iPSCs to identify whether any rescue cell ...CRISPR plasmids to generate deletions of candidate genes in euploid iPSCs. “The BeHEARD Addgene reagents ...

...variety of genes with GFP. These plasmids are available through Addgene, and the stem cell lines we’ve made...is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure...information and images generated from these cell lines can be found in the Cell Catalog on our data portal...different human cell types, and while most of the genes we’ve tagged are widely recognized markers of cellular...initial set of 10 plasmids representing 10 human genes and will add more to the collection later this year...everywhere To find out more about gene edited hiPS cell lines generated using the Allen Institute for Cell Science...collection includes systems for tagging mammalian cell lines, Drosophila cells, and C. elegans. In addition to...

...fluorescent proteins, and luminescent proteins. While fluorescent proteins and luminescence have been widely used...maturation times, and fitness cost of expression. They found that the cellular fitness cost varied greatly...plasmid that combines the most desirable features: intense color, fast maturation, and low fitness cost yet...striking visuals from fluorescent jellyfish, bioluminescent phytoplankton, and colorful corals, to name...instrument-free detection. Unlike fluorescence or luminescence, which require UV lamps, fluorometers, or luminometers...3: pTOX plasmids expressing amilCP and tsPurple genes. Image: Lazarus et al., 2019. One such lab is...