... SELEX could be used to classify DNA aptamers as well, and over the course of the next two decades, these..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible...with such techniques can be problematic, however. Well designed aptamers can help reduce such background... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting temperature...cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further enhance...

...expanded since its adaptation for use in mammalian cells in 2012-2013. Researchers are now using this system...successfully adapted for use in screening experiments as well). Therefore, if you’ve started down the CRISPR path...500bp is best) and select the organism. It will then tell you the available gRNA targets in that region. The...plasmids containing sgRNA sequences into HEK293T cells. The fluorescence intensity was classified into ...is then a simple matter of transfecting HEK293T cells with your modified pCAG-EGXXFP plasmid along with...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ...you’ve validated your gRNAs for use in mammalian cells, it’s time to use them in mouse embryos. When researchers...

...to reproduce itself as it must to survive within cells. The replicons of plasmids are generally different...sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs are held together with two hydrogen...’s sake, we've ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria...pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and 15A. Not all...example, the pMB1 ori maintains about 20 copies per cell, while pUC – which differs by only two mutations...mutations – will produce as many as 700 copies per cell. Figure 1: A plasmid map showing the standard ...positives? Will you have only one plasmid type in your cells at a time? Do you want to make a lot of your plasmid...

...expressing target cells. Any neutralized pseudoviruses would be unable to get into the target cells and this can...Khan K, Bernstein M, Balazs AB, Gosnell BI, Hanekom W, Moosa M-YS, Lessells RJ, de Oliveira T, Sigal A (2021...disease severity and survival. Cell 184:476-488.e11. https://doi.org/10.1016/j.cell.2020.12.015 Garcia-Beltran...vaccine-induced humoral immunity. Cell 184:2372-2383.e9. https://doi.org/10.1016/j.cell.2021.03.013 Additional resources...variant), B1.1.298, and B.1.429 (Epsilon), nearly as well as they could for the wild-type. However, variants...vaccines and allows the virus to gain entry into human cells via human ACE2 receptors. In their assay, the pseudoviruses...

...coordinated by the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation...avoiding toxic intermediate buildup as well as controlling cell fate decisions. By using an orthogonal...or metabolism. Our attempts to manipulate these cellular behaviors often fall short with the generation...bottlenecks and their scope is limited by the lack of well characterized inducible promoters. Far removed from...for sequence-specific control of gene expression. Cell, 152(5), 1173-1183. (2013). PubMed. Zalatan, J...Transcriptional Programs with CRISPR RNA Scaffolds. Cell, 160(1), 339-350 (2015). PubMed. Find the Plasmids...crispr/qi/ Plasmids described in  Zalatan et al. Cell  (2015) can be found here: https://www.addgene.org...

...CRISPR-Cas Immunity. Cell, 161(5), 1164–1174. https://doi.org/10.1016/j.cell.2015.04.027 Stella, G., & Marraffini...and is able to target and edit RNA in mammalian cells. Type IV Type IV is the middle child (like me!) ...and more. Type II Type II is the most popular and well-known type, as this is where Cas9 falls. The mechanism...mechanism of type II is, unsurprisingly, well understood, and the first taught when introducing CRISPR ...., Belanto, J. J., Verdine, V., Cox, D. B. T., Kellner, M. J., Regev, A., Lander, E. S., Voytas, D. F....Suberski, A. J., Mears, K. S., Demircioglu, F. E., Moeller, L., Kocalar, S., Oshiro, R., Makarova, K. S., ...Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing...

...path.   Yeast as a model organism Yeast are single cell, eukaryotic organisms formally classified as fungi...and S. cerevisiae (budding). Yeast make for an excellent model organism for a few reasons: Affordable...yeast? It’s actually not new at all, but it’s not as well-known in the general research community. Phillips...methanol as its sole source of carbon and energy (as well as glucose or glycerol). K. phaffii is only distantly...similarity to the ancient yeast strains and metazoan cells.   Applications for K. phaffii There’s a number ...protein bang for your media buck - it grows at higher cell densities than most of its counterparts, yielding...studies Peroxisomes are small, membrane-bound organelles essential for sequestering certain enzymes and...

... of all of the mRNA bound-ribosomes in a cell. When cellular mRNAs are digested with ribonucleases, ribosomes...Regulating translation is key to cellular function, especially during development or stress. With ribosome...explore the complexity of translational control in cellular physiology. Ribosome profiling Ribosome profiling...the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable this...are bound to the reporter, causing it to appear yellow. The act of translation pushes NLS-PCP-GFP off ...the effects of 5’ UTR variants in translation of cell cycle protein Emi1, showing that ~80% of transcripts...powerful tools for studying translation in various cell types and physiological states. References 1. Ingolia...

...plant protein TIR1 in non-plant cells, which interacts with the cells’ endogenous ubiquitin ligase, the...able to translate AID to yeast, as well as mammalian and human cell lines (Nishimura et al., 2009, Natsume...degradation using AID Article contributed by Alyssa Cecchetelli Auxin Inducible Degradation (AID) uses plant ...based method to tag endogenous proteins in HCT116 cells with mAID. Using this method, the lab was able to... protein of interest to create conditional human cell lines. Lastly, the Kanemaki lab demonstrated that...the target sites adjacent to an NG PAM in human cells. The increased versatility of the engineered SpCas9... just a 2-D linear molecule snaking through our cells’ nuclei--our chromatin is comprised of thousands...

... can rupture E. coli spheroplasts (cells which have had their cell walls removed with lysozyme). This ... blogger Jessica Polka, a Postdoctoral Research Fellow with Pamela Silver.  Most types of biological motion...distance? And can we understand such a structure well enough to engineer it to suit our purposes? In ...distance? And can we understand such a structure well enough to engineer it to suit our purposes? These...protein fusions allowed us to image R bodies inside cells, and these constructs may be useful to future scientists...applications. For example, you could use them to lyse cells (and surrounding membranes) in keeping with their...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...

...patterning of cells and tissues is fundamental to development. I'm interested in how cells find their way...competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/corresponding...quality journals. It helps to come from the lab of a well-known investigator. If you only have middle author...minimize jargon. Equally importantly, you really are selling yourself. A sales pitch that requires reading two...that I had a flair for communication was rapidly dispelled. She would ask me, "Is this what you mean? Are... foot in the door. Your ability to interview and sell a vision for your contribution to a Department's...remember the days of modest postdoc salaries all too well. If there's a group to make money from, postdocs...

...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...joined to fluorescent molecules are applied to a cell or tissue sample where they bind to their targets...to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case of IHC...concentration and incubation time for this step as well. Preservation and Imaging Similar to Sample ...

...and beads that are unable to bind to them: like cells but with far more predictability. The beads create...The benefit of beads is that they do not require cells from your experiment, and they are non-specific....flow cytometer, you generally want your positive cells to read between 104 and 105 in order to have enough...the plot to see all of your positive and negative cells. This can be achieved by adjusting your voltage ...of your experimental samples, combine it into one well or tube, and stain it with every marker you are ...gating schematic to easily determine where your cells of interest are (B).   Using FMO controls In...markers or commonly used markers that define broader cell populations (e.g., CD4, CD8b, CD11b, CD19), are ...

...expressed in cells that express cre recombinase. Typically, this feature is used to attain cell-specific ...CRISPR applications and can be expressed from your cell line of choice after transduction with this virus...blast virus can be used to generate Cas9-expressing cell lines, which can then be used both for screens (...viral prep to create a single stable Cas9-expressing cell line and then transduce this line with lentiviruses...using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this growth rate effect doesn’t ...activating particular signaling pathways in specific cell types (typically neurons). While all of these viruses...transduction and transgene expression across different cell types (Aschauer et al., 2013). Expression of hM4D...

...fundamental approach to studying membranes, organelles, and other subcellular compartments is via proteomics--the...gene knockdown Article contributed by Alyssa Cecchetelli  Listen to the C. elegans RNAi podcast segment... the gene being targeted via RNAi. Recently the Vellai lab has created an improved RNAi vector that leads...incorporated into the dsRNA necessary for RNAi. The Vellai lab’s new plasmid, T444T, contains T7 terminators...interaction networks of specific proteins in living cells. Until recently, the enzymes most frequently used...but requires H2O2, which can be toxic in living cells. BioID was derived from biotin ligase and uses far...detect viral outbreaks Article contributed by Izzy Mueller  Listen to the viral detection podcast segment ...

...activity. These cells, presumably the cell you want, cannot break down X-gal and are white. Cells that don’t...screening strategy as well. In a screen, you aren’t killing off a portion of the cells like in a selection... the plasmid you’re looking for out of the many cells that don’t. One way to begin the search is by using...using selection strategies, where only cells that have gained or lost a specific gene survive (ex: antibiotic...selection. Instead, all cells survive and you will need to sort through them to find the desired clone. Do your...bacteria that have taken up this vector, but it won’t tell you whether the vector contains the gRNA insert ... plates containing X-gal allows you to identify cells that took up the vector and distinguish between ...

...sequencing studies (as well as reference cDNA clones mapping to well-annotated cellular pathways). It wasn...gene of poorly classified function and a well established cellular signaling gene, thus providing a starting...sequence-confirmed series of clones corresponding to known cellular signaling pathways as part of our Genetic Pathway...Mutations. Berger AH, Brooks AN, Wu X, et al. Cancer Cell, 2016. PubMed PMID: 27478040. The lung cancer mutant... profiling of human gene and allele function via Cell Painting. Rohban MH, Elife, 2017. PubMed PMID: 28315521...functional assays for these mutations and map other cellular functions and phenotypes. Since time is of the... of MAPK1/ERK2 Missense Mutants. Brenan L et al. Cell Reports, 2016. PubMed PMID: 27760319. PubMed Central...

... protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of...disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant...Assembly Cloning and Gateway® cloning technologies, as well as several others. Each method is unique and relies...which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB...broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon...Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly...can one work with a plasmid/gene that kills the cells expressing it? The answer lies in special strains...

... engineer cells for specific purposes. Unfortunately, plasmid copy numbers vary within cell populations...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes.... When cells are dividing rapidly, multiple rounds of replication are initiated before the cell has divided...growth rate can all impact plasmid copy number in a cell. One can attempt to eliminate fluctuations due ... pSC101 with copy numbers ranging from 3-100 per cell, a stabilized promoter buffered against the effects... to stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold...fold (for p15a, R6K, and ColE2 plasmids) as cell division slows (Kittleson et al., 2011). Segall-Shapiro...

...through programming cells. In the context of therapeutics, if you want to program a cell to identify and ...translational activity a cell has and control how much it taxes the resources of a cell. The objective is to... to the regulatory networks within E. coli host cells. Image from Nielson and Voigt (2014). ...within the wood. There are different types of cellulose fibers, pattern, shape. All of that is controlled...and target a malignant cell, then you’d need the ability to turn on genes at different times. It requires...being used. One thing is that Cas9 is toxic. When cells carry CRISPR circuitry, it can slow their growth...combination of parts in Addgene along with the software to tell you which parts are required to get the function...

...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...best to be well-rested and alert the day of the procedure. Mice move much more quickly than cell cultures...become a regular part of basic biological research as well as clinical therapy. These biological tools are ...re derived from viruses that can infect people, cells, and animals. Some of these viruses are completely...best practices for handling lab-grown viruses as well as potential risks and how to minimize them. Built-in...While the virus can still enter (and thus infect) cells, this safety feature prevents the virus from replicating...replicating and spreading in non-target cells, including yourself as the researcher.   Figure 1:...

...Davidson AR, Maxwell KL, Doudna JA (2017) A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell 170:1224-1233...Sontheimer EJ, Maxwell KL, Davidson AR (2016) Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167:1829...concern is that the longer Cas remains active in a cell, the greater chances there are for off-target edits...Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system, N. meningitidis can also ...within the first six hours. When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4...1233.e15 . https://doi.org/10.1016/j.cell.2017.07.037 Li C, Psatha N, Gil S, Wang H, Papayannopoulou T,...CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells. Molecular Therapy - Methods & Clinical Development...

...stain the cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue...specific cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the...techniques to alter gene expression or to label specific cells, but there are too few resources to teach students... required for both WISH and RNAi experiments, as well as how to perform worm amputations. They also created... in tissue. This is useful for labeling specific cell types and for viewing how gene expression changes...modified nucleotides (for example, Digoxigenin-labelled Uracil) that will bind to complementary mRNA within...Williams, Eric J. Ross, Sofia M. C. Robb, Sarah A. Elliott, Kimberly C. Tu, Alejandro Sánchez Alvarado, The...

...fluorescent proteins available. Fortunately, several excellent reviews have collated data from various papers... monitoring sensitized emission in living plant cells." (2013). PubMed PMID: 24194740. PubMed Central ...source of error in suboptimal or undetectable FRET, well-characterized FRET reference standards can be used... alters the FRET efficiency. Detecting certain cellular changes with an intermolecular probe is often ...Miyawaki et al., 1997), was designed to measure intracellular calcium and published in 1997. Since that time...of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments can probe...concentration or activity of small molecules, detect cellular processes and signaling cascades, quantify mechanical...

...Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories...purpose and mechanism of polyadenylation vary across cell types, but polyadenylation generally serves to promote...naturally occurring terminators, such as T7 and rrnB, as well as engineered high-efficiency terminators such as... thought that poly(A)s are used to control the cellular concentration of regulatory RNAs and may additionally...additionally act as a quality control mechanism to rid the cell of mis-folded RNAs. Eukaryotic termination and polyadenylation...transcribes tRNA and other short RNAs. Although not as well studied as prokaryotic termination, the basic processes... the nuclear export and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation...

... primary cells. The authors used this platform to assay dozens of GPCRs across many cell types in...biosensor platform. Cell, 187(6), 1527–1546.e25. doi: https://doi.org/10.1016/j.cell.2024.01.028. New...Prime editing (PE), initially developed in human cells, has been uniquely challenging to apply in dicotyledonous...labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then used single-molecule...constructs for tagging the kinesins KIF5A and KIF5B as well as backbones for generating other SNAPf-, CLIPf-...CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids will be useful not only for...recordings of spontaneous activity in human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured...

...infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface receptor...antigenic drift.    Starr et al Cell. 2020. https://doi.org/10.1016/j.cell.2020.08.012  Starr et al Science...Library generation and sequencing. From Starr et al Cell., 2020. (B) Mapping antibody-escape mutations with... SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface. The Bloom lab used the library to identify... candidates were validated by IP-MS using HEK293 cell lysate where the antigens would be in their native...immunoprecipitate the entire complex in three different cell lines. The Gräslund lab plans to continue their ...protection in yeast bioproduction pathways By Gabrielle Clouse Claudia Vickers’ lab has created a novel...

...Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria. Since that discovery, the family... localization of that biomolecule inside living cells. FPs classified by the emission color (emission ... the variants blue FP (BFP), cyan FP (CFP), and yellow FP (YFP) were derived. For a breakdown of GFP, ...467 nm Cyan 474 - 492 nm Green 499 - 519 nm Yellow 524 - 538 nm Orange 559 - 572 nm Red 574 - .... Therefore, these can be used as “timers” for cellular processes following their activation. The four... longer-wavelength filters (e.g. GFP also emits yellow light). Note that some combinations of FPs can ...Guest Blogger! Gal Haimovich, PhD, is a research fellow in the lab of Prof. Robert Singer at Albert Einstein...

... Ba/F3 and MCF10A cellular models. Ba/F3 and MCF10A are growth factor dependent cell lines and any mutations...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic ...corresponding wild-type genes were constructed as well. They called this library the CTD2 FASMIC Plasmid...functional groups, based on how each one impacted cell viability (see figure). Most of the mutations were...to be “neutral,” having no significant effect on cell viability compared to a wild-type control. Approximately... the mutations, however, significantly increased cell viability, and these “activators” may be the direct...represented in this collection along with hundreds of less well-studied candidates. The possibilities are vast. ...

... are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct...shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research...turnover rate. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would...made by accident. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. ...excitation at 561 nm. This imaging process worked well with the common GFP variants EGFP, mGFP4-T, and ...spontaneous conversion did not occur. Tobacco suspension cell culture with cytosol-targeted GFP. White circles...this technique may not be appropriate for high-pH cellular compartments. Sattarzadeh et al. have shown that...