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... 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...

...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...

...also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both...self-labeling protein fusions as well as empty backbones to clone your own protein of interest. Fluorescence...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...maintained through mechanisms like fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like...for super-resolution usages, such as superior brightness, photostability, and most importantly, a photoswitching...combination is Alexa594, and Alexa647 due to their brightness and high degree of overlap. Dyes for DNA/RNA ...The field of fluorescent ligands and dyes has witnessed remarkable advancements, driven by the need for...

...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...

...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...

...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...

...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) ...

...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...

...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...

...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...

...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...

...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...

...field in which covalent modifications to DNA or histones, the proteins that help package DNA, were associated...active or silent, they could identify the target genes of previously uncharacterized enhancers. The popular... Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter and enhancer regions. These marks ...lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation...lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation...a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology... DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nature...

...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...

... and high binding capacity. In addition, PVDF membranes can be repeatedly stripped of an antibody and ...antibodies used for staining. To address this, the membranes are incubated before staining in a buffer containing...across tissues. Recently, knockdown or knockout lines have become the gold standard in antibody validation...(Pillai-Kastoori et al., 2019). Tissues or cell lines other than those used by the vendor for validation...controls. Positive controls typically include cell lines or tissues that are known to express the protein...specific substrate. The emitted light, or chemiluminescence, is detected on x-ray film or with specific...standards.  Protein abundance In addition, the thickness of the band provides information about the relative...

...research, including over 4,000 cell lines. Seventeen of these cell lines were used to investigate the susceptibility...repositories that distribute mouse strains, plasmids, cell lines, and much more have been critical for pandemic research...Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate SARS-CoV...research into this virus. One such example is cell lines. The American Type Culture Collection (ATCC) is ...additional cell line used in this study came from the Japanese Collection of Research Bioresources Cell Bank,... pathogen. The sharing and distribution of cell lines from repositories on opposite sides of the world...

...vivo research and the CARE guidelines for case reports are two sets of guidelines frequently used by editors...journal, it is important to check the journal’s guidelines to authors and see whether your paper is in scope... scope for the journal’s coverage. These guidelines also provide information on a journal’s house style...use to the scientific communities. The ARRIVE guidelines for the reporting of laboratory animals during...interest of the journal. Be sure to check the Author Guidelines of the journal you are submitting to and make... your paper. Always check the journals Author Guidelines to make sure that your paper fits with the journal...

...of traditional cloning is the identification of clones that actually contain your insert of interest. ...of the gene and how molecular biologists have harnessed it to evolve cloning technology. CcdB: A potent..., leading to positive identification of desired clones.   ...Becomes a powerful cloning tool Molecular... 20 years ago and developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained...This procedure dramatically reduces the number of clones that do not contain the recombinant plasmid and...investigator’s insert of interest. Hence correct clones are identified much more efficiently, as those ...propagate and prep your ccdB-containing empty backbones but should never be used when selecting for recombinant...

...interested in heftier genes, you can coinfect with multiple viruses to express genes or gene sets over 4.7...expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged in them and...icosahedral capsid, that can be used to express genes of interest in cell and animal models. AAVs are ... their genomes “gutted”, meaning that all viral genes except ITRs (inverted terminal repeats – the sequences... 4.7 kb, meaning that your gene of interest (or genes) need to be 4.7 kb or less to fit in a single rAAV...

...Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental manipulations...saving the time and money needed to develop new lines. Using nanobodies to create GFP scaffolds for transcriptional...but knew it would take years to develop the mouse lines required for this purpose. Tang wondered if he could... could use previously established mouse lines expressing GFP in specific cell types to do more than just...then tested T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells with GFP...optogenetics, opening up the possibility of adapting GFP lines for optogenetics experiments in specific cell populations... open up many new possibilities for GFP-labeled lines, and they each have their own advantages. T-DOGGs...