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We narrowed to 902 results for: nes

Showing: 261 - 280 of 902 results

  1. Evolution of Lab Techniques

    Type
    Blog Post
    ...the location of genes. With this knowledge, researchers were able to target specific genes for deletion ... at Harvard University. Just as computers, cell phones, and cars become more technologically advanced ...fluorescently labeled probes. Colorimetric and luminescence based detection methods are also commonly used...ddNTP, and then run these reactions out in separate lanes of a gel. Since the sequencing gel will separate...nucleotides were replaced with fluorescently labeled ones allowing for fluorometric-based detection. This ...generally considered first generation DNA sequencing machines (9). Today, similar technologies, sometimes referred...make techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/...

  2. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    ... our purposes? In search of simple biological machines R bodies can repeatedly extend and contract...using R bodies: simple, force-generating protein machines that can extend and retract over many microns ...with proteins and remain functional, 2) break membranes outside of their natural context or 3) be modified...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...demonstrated that R bodies can be “tuned” with mutagenesis to extend at different pH values. Using a screen...in a particular region of the R-body. Further mutatgenesis of this region produced R-bodies that could ...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...

  3. Working with Nuclear Receptors

    Type
    Blog Post
    ... disease, cholesterol gallstone disease, senescent illnesses, and the metabolic syndrome. Coregulators...functional groups of genes involved in the execution of a specific regulatory regime (such as genes involved in...ligand-dependent sensors for a diverse set of fat-soluble hormones, vitamins, and dietary lipids. Included in this...remain unknown. Together, NRs govern expression of genes involved in a broad range of reproductive, developmental...For example, the phosphorylation status of SRC-3 defines its association with other members of the complex...as in important physiological states such as adipogenesis and macrophage activation. Our mass spectrometry-affinity...

  4. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...2014). Microbial rhodopsins are encoded by opsin genes, and include bacteriorhodopsins, halorhodopsins,...control the flow of electric charges across cell membranes and maintain membrane potential in response to...light-sensitive, so the selective expression of opsin genes in targeted cells makes it possible to control neuronal..., 2014). This could be overcome by fusing opsin genes to cell-type specific promoters; however, those ...those usually drive weak expression of downstream genes. Strong cell-specific promoters, on the other side... circuitry behind the mechanisms of sleep and anesthesia. In Cre transgenic mice, they targeted the dopamine... previously subjected to steady-state general anesthesia (Taylor et al., 2016). These very same neurons...

  5. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    .... These could be genes, specific polymorphisms, including antibiotic resistance genes. This approach successfully...Genome editing tools for bacteria CRMAGE system combines CRISPR and recombineering-based MAGE The Nielsen...CRMAGE system is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex...Yang’s lab describes a two-plasmid system that combines recombineering with CRISPR to create a system ...effectively silenced transcription of one or more genes in E. coli. and in a mammalian cell line (Qi et ... repressed or activated transcription of select genes in E. coli. Furthermore, to demonstrate wider applicability... the Husson lab used CRISPRi to study essential genes in M. tuberculosis. dCas9 and a gRNA are expressed...

  6. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

    Type
    Blog Post
    ... and bleed dates and even clonal hybridoma cell lines can produce more than one mAb (Bradbury et al., ...Hybridomas can lose gene expression of the mAb-encoding genes or fail to revive after cryopreservation (Bradbury...antibodies that are generated in vitro using synthetic genes that are typically expressed from a plasmid or 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...

  7. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ... AdVs are stripped of regulatory genes E1 and E3. Without these genes, AdVs cannot replicate on their ...produced at high titers, can accommodate large transgenes, transduce quiescent and dividing cells, and ..., or high-capacity (HC-Ad__ are devoid of viral genes and therefore can accommodate up to 36 kb. Gutless...can be produced in E1-containing mammalian cell lines such as HEK293 cells. 1st generation AdV cloning...immunogenicity. In addition to E1/E3, non-structural genes E2 and E4 are deleted in 2nd generation AdVs, thereby...cloned) and pAdEasy™ which contains the adenoviral genes necessary for virus production. For detailed instructions...therapies in the new era of personalized medicine." Genes & diseases 4.2 (2017): 43-63. PubMed PMID: 28944281...

  8. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila through homologous...techniques and the plasmids used to inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based... Neomycin, puromycin, and hygromycin resistance genes are commonly used for positive selection. While ...methods have been used to create many knockout cell lines and animal models, their efficiency is very low,...easier to use and are more efficient at inactivating genes. CRISPR can target a genomic sequence and create...perfected, the production of cells and mouse knockout lines should increase. New genome engineering tools also...have tools for creating knockout animals or cell lines, please consider sharing them by depositing with...

  9. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    ... Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or ...cells or a selection marker to generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA...contains a selection marker to generate stable cell lines. Packaging and Envelope plasmids provide the necessary...and/or gRNA in a wide variety of mammalian cell lines. Useful for difficult to transfect cell types and... cell.   Delivery methods for mammalian cell lines As mentioned above, the expression system you choose...Delivery of these components into mammalian cell lines is quite broad and includes several different methods...

  10. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry... Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet...negative” photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't...background fluorescence, but their single molecule brightness and ability to switch from a dark state to a ... Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) Dronpa Monomer UV-Violet...there are several parameters to consider, such as brightness, photostability, pH stability, chromophore maturation... is the contrast ratio- a comparison of PA-FP brightness before and after photoactivation. A high contrast...

  11. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ...binding. Formaldehyde cross-links the protein (red lines), which maintains structural epitopes (orange region...proteins that need a little help crossing cell membranes as they are not able to diffuse across natively...Permeabilization essentially punches holes in the cell membranes of your sample to allow antibodies better access...example, saponin interacts with cholesterol in cell membranes and leaves many membrane-associated proteins in...detergents that interact with lipids and proteins in membranes and permeabilize non-selectively. This non-selectivity...helpful when trying to pass tough-to-permeabilize membranes and these detergents avoid some of the negative...but it is better at permeabilizing organellar membranes. Created with BioRender.   How do I choose...

  12. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    ...substantially enhance the brightness and photostability of the classic rhodamines and their analogs without... a greater burden on the photon budget, which determines how long and precise we can track individual ...selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and the complexity... classic small molecule fluorophores, such as rhodamines (Figure 2a), were discovered in the 19th century...due to its established chemistry, small size, brightness, and cell permeability. In past decades, further... the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless...PMCID: PMC4344395. 3. Lavis, Luke D., and Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical...

  13. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    ...PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the Deverman and Wilson labs determined...as non-permissive to PHP.B CNS transduction. Two genes were associated with the PHP.B permissive phenotype... suggesting it was a likely player in PHP.B permissiveness. The Wilson Lab also looked for the gene variant...mice to map the inheritance pattern of PHP.B permissiveness. The first generation of outbred mice (F1) ...~4.5 Mbp region of the mouse chromosome. Of the genes located in this stretch of DNA, Ly6a seemed the ...cells: galactose and the AAV receptor (AAVR). Cell lines lacking galactose were still transduced by PHP.B...to the CNS. The bottom line of viral vector permissiveness While PHP.B has impressive CNS transduction...

  14. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...Toth) These wasps are like the ones that nest on your house. Their nests are open so you can see them and... human interaction for all of us studying human genes in test tubes and gels). As a freshman in college... I studied development and evolution of histone genes in Strongylocentrotus purpuratus, the spiny purple...silencing in the wild. They also return to their nests so can be studied over time. They recognize individual... but the Gorbsky Lab is using Xenopus frog cell lines to develop robust methods for frog gene editing.... DNA at all (maybe because they have only 1,700 genes total). Hydra (Celina Juliano) This is a known stem...

  15. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    ...varies across targets, they tested two genes in mice. For both genes, they saw indel formation and phenotypic...certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an...al. successfully used CjCas9 with AAV to target genes in mouse muscle and eye tissue. Neisseria meningitidis...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes...ncomms14500  Levy JM, Yeh W-H, Pendse N, Davis JR, Hennessey E, Butcher R, Koblan LW, Comander J, Liu Q, Liu...https://doi.org/10.1002/biot.201400046  Strecker J, Jones S, Koopal B, Schmid-Burgk J, Zetsche B, Gao L, Makarova...

  16. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ...sequences While PAM sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the human...circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the...Cas9 homologs derived from species other than S. pyogenes, and 3) the use of non-Cas9 enzymes. (For more...check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel PAM recognition In 2015, Keith... screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to cleave target DNA sequences...variant is far more selective for the canonical S. pyogenes PAM sequence NGG compared to wild-type SpCas9,...Cas9 species PAM sequence ’) Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) ...

  17. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...cancer drug targets by finding genes that cause cell death in cancer cell lines upon activation (Gilbert et...for specific gene targets CRISPR activation for genes with known functions has the potential to be used...et al., 2015) into neuronal cells by activating genes that cause differentiation and to reverse HIV viral...., 2015)) are good choices across multiple cell lines (HEK293T, MCF7, U2-OS, Hela, N2A, 3T3) and organisms...E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon...Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, Haque SJ, Cecchi RJ, Kowal EJK, Buchthal J,...Structure, Function, and Membership Using Genome-Scale Fitness Screens. Cell Systems 6:555-568.e7 . https://doi.org...

  18. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...al. "Epitope tagging of endogenous genes in diverse human cell lines." Nucleic acids research 36.19 (2008...endogenous alleles of the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also ...cells. After G418 selection, individual gene edited clones are identified by PCR. Then, if necessary for subsequent...knockin efficiencies of 1-40% with G418-selected clones. However, there are important disadvantages to ... of a gene, or which require modifying multiple genes simultaneously in the same cell, are more appropriate...AAV-based approach requires G418 selection of infected clones, making it unsuitable for most in vivo applications...using adeno-associated virus (AAV)." Chromosomal Mutagenesis. Humana Press, New York, NY, 2015. 75-103. PubMed...

  19. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

    Type
    Blog Post
    ...mixture of XFP genes. Stochastic multicolor labeling is possible when each of the XFP genes is introduced...express one of the three fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene...result in a combinatorial expression of these three genes with different copy numbers, producing dozens of...et al., 2007; Cai et al., 2013). However, the brightness of Brainbow was inherently low. This is because... Plasmid concentrations and AAV titer - The XFP genes have to be introduced at ~2 copies per cell per ....7166 (2007): 56. PubMed PMID: 17972876. Kohl, Johannes, et al. "Ultrafast tissue staining with chemical...

  20. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ...target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant... GAL4/UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter...necessary or desirable to control or look at multiple genes at the same time.  The 3 binary systems listed above...Drosophila or for precise control over multiple genes in synthetic systems. Recent work has even used ... simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718...and interconvertible Drosophila overexpression transgenes. Sci Rep 12, 3835 (2022). https://doi.org/10.1038...Sneha Srikrishnan. “Introduction and expression of genes for metabolic engineering applications in Saccharomyces...
Showing: 261 - 280 of 902 results