...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...

...Funded by the NSF (with Co-PIs Jessica Bell, Lisa Gentile, Ellis Bell, and Amy Springer), this group now ...Poirier, V., Pellettieri, J. 2020. The exon junction complex is required for stem and progenitor cell maintenance...identifying new genes required for stem and progenitor cell maintenance in planarians in Developmental Biology...diversity and inclusion in STEM disciplines (Bangera & Brownell, 2014; Duboue et al., 2022), as they increase ... his teaching style, with the former offering excellent training in improvisation and the latter making...CURE trainee goes on to win a Nobel!   Jason Pellettieri is Professor and Chair of the Biology Department...References and Resources References Bangera, G., Brownell, S. 2014. Course-based undergraduate research ...

...functional in cultured Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the ...recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo...standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion...activity and specificity with low cross-reactivity, as well as low toxicity. FLP/FRT recombinases (from Saccharomyces...recombination efficiency. When expressed in neuronal cells, each line showed broad recombinase-dependent RFP...these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to be functional... damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak B, ...

...optical labeling and tracking of proteins, organelles, and living cells in a spatiotemporal manner, which is...eight millimeters – not single cells (yet) but small clusters of cells. Looking ahead What’s next, you...fluorescent proteins to observe what’s happening inside cells for at least a couple of decades. Green is the classic... were lots of fluorescent proteins made from jellyfishes and corals, but all of them fluoresce outside... vivo. “One of the reasons we can’t localize one cell at a depth of one centimeter with fluorescence is...on the other hand, are larger than the size of a cell, so they scatter much less. As a result, ultrasound...brighter, more photostable, smaller, and less toxic to cells. They’d like to make near-infrared fluorescent proteins...

...if the cell dies from injury, this protein leaks out into the cytoplasm, migrates out of the cell, and ...Keytruda) causes T-cells to more easily recognize and cause the destruction of cancer cells. Pembrolizumab...cause inflammation, which can cause mesothelial cells to mutate and ultimately form cancerous tumors.  ...resection), systemic chemotherapy to kill cancerous cells that may have spread beyond the point of origin,...diagnosed so late that these treatments do not work very well. The best option in such instances is palliative...successful in killing many (if not all) of the cancer cells that may be left behind after surgery, while being...and binds cells that can begin an immune response. This important biomarker can be used to distinguish between...

... Guide RNA and Target DNA. Cell 156:935–949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong ... Delivery by AAV Vectors. Cell 181:136–150 . https://doi.org/10.1016/j.cell.2020.03.023 Additional Resources...acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels at a similar efficiency ...package each piece into an individual AAV. When a cell is transduced by both AAVs, the full length gene...SpCas9 which had modest editing rates in HEK-293T cells when compared to the full-length SpCas9. In 2016... DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing with CRISPR-Cas13... allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: ....

...low-frequency cancer mutations from cell-free DNA fragments as well as health-related single nucleotide...Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Additional resources...similar types of HPV, HPV16 and HPV18, from human cells, at atomolar levels, within one hour. DETECTR thus...science.aaf5573 Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados ...10.1126/science.aam9321 Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (2018) Multiplexed...Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF, Barnes...of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https://doi.org/10.1016/j.molcel.2015.10.008...

...when working with cell culture, because it is easy to harvest large quantities of cells. However, when working...numbers of cells or when a high degree of signal to noise is desired. It also provides an excellent additional... been widely applied in many model organisms and cell lines. Although ChIP-seq is a relatively simple ...typically requires a large amount of input material, cells or tissue, to produce a strong enough signal over...protein of interest. First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic...the usage of a strong detergent to permeabilize cells rather than to rely on nuclei extraction. They report...technique, specifically in terms of initial steps of cell or tissue preparation.   All in all, CUT&RUN offers...

...to infect and replicate in host cells, but they are susceptible to cells’ defense mechanisms. The use of... transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double strand break...improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB...tranposase variants for hyperactivity in mammalian cells. Their efforts resulted in a new and improved SB...of antibiotic-resistance genes conducted in HeLa cells). In their Nature Genetics paper, the lab showed...allows the rapid generation of stable transgenic cell lines which can robustly express your gene-of-interest...Beauty transposons rapidly generate stable transgenic cell lines. Biotechnol J. 2015 Feb 4. doi: 10.1002/biot...

...transformation of healthy cells into melanoma cells. They’ll combine induced pluripotent stem cell (iPS) technology...gene appears to play a role in many cell processes involving cell membrane dynamics. When they learned...still no treatment available. Aguilar is a basic cell biologist. He got interested in Lowe Syndrome initially...the gene, it piqued their interest. They obtained cells from patients with the condition to reveal some ...explore the effect of various OCRL1 mutations on cell behavior in greater detail. The goal is not just...now plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models carrying different SOX10...

... to express one gene in one cell type and another gene in a different cell type (6–8). Although it’s often...systematically study gene function with temporal control and cell-type specificity! The Gal4/UAS system At a more...the hedgehog gene can be overexpressed in several cell types by using different Gal4 drivers. It is much...much easier to mate flies to get the right cell type expression as opposed to making a new fly with hedgehog... Targeted gene expression as a means of altering cell fates and generating dominant phenotypes., Development...PubMed PMID: 15262411. 4. T. D. Southall, D. A. Elliott, A. H. Brand, The GAL4 System : A Versatile Toolkit...Neuro-Trauma during Asymptomatic Viral Infections, Cell Host Microbe 22, 48-60 (2017). PubMed PMID: 28704652...

...non-growing cells are orange, slow growing cells are orange-green, and fast growing cells are green. A...fluorescence. Cells expressing the library are then sorted by fluorescence-activated cell sorting into ...for serotonin. According to Unger, “serotonin is a well-known but poorly studied neuromodulator, and evidence... A second colonizer, labelled with a constitutively fluorescent reporter can also be used to track how...population. Last, Bernach will simultaneously monitor cell cycle and growth rate using TIMER and an unstable...reporter under the control of promoters that control cell cycle gene expression.  Additional Resources on...

...aiming to dedifferentiate mouse brain cells into neural stem cells. I was quite unsuccesful (although I... work in sociology, behavior, and artificial intelligence (including a team that created a novel form ...game Go). In addition, there was an entire story-telling session organized by Story Collider and which featured...neuroscience trade. There were, of course, many companies selling antibodies, microscopes, and dissection tools, ...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 networks, and...Addgene booth to learn more about these tools and to tell us how useful the service has been for their experiments...

... shared. Can you tell me a bit about your job? Katz: I’m the Director of Intellectual Property Programs... Board of Directors. Katz is the Director of Intellectual Property Programs at Suffolk University Law ...provides interesting and unique challenges for intellectual property lawyers. While there will always be...there will always be an interest in protecting intellectual property. Figuring out that balance is, and ...Suffolk is to help create new programmatic content as well as help our students succeed whether that be academically... Karen Katz, Esq Ms. Katz is the Director of Intellectual Property Programs at Suffolk University Law ...she was on law review. She clerked for Juan R. Torruella, U.S. First Circuit Court of Appeals. She served...

...plasmid. Envelope proteins determine which types of cells a virus can infect. The VSV-G envelope protein enables... enables this virus to infect many different cell types so it is useful in a wide range of applications... The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR v2...deliver Cas9 and your gRNA of interest into mammalian cells. This vector was the top requested plasmid of 2015... popular in genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using...puromycin cassette that makes it less efficient in some cell lines. pSpCas9(BB)-2A-Puro (PX459) V2.0 was made...capable of inducible expression of shRNA in target cells by the addition of tetracycline/doxycycline to the...

...spin down the samples to remove cell debris, which will collect in a pellet. The supernatant will contain... you may be thinking, well, these are mostly fairly standard molecular and cell biology techniques. What... Process ChIP can be done for either tissues or cells. The process is very similar for both, though tissue...Both polyclonal and monoclonal antibodies can work well in ChIP. If you’re using an antibody for the first...What’s all the fuss? Well, ChIP’s complexities come not from the techniques used, but from the difficulty...will also have to be optimized for both tissue or cell type, amount of protein, and desired fragment length...other antibody applications, this investment can well be worth it once you generate your data. However...

... plot would serve you well. Percentage of cells in a parent gate (all live cells for example) that are...percentage of total live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive ...example, you have introduced a GFP protein into your cells (visualized via the FITC channel) and you are trying...graphs could simply be the percentage of the total cells present in the gate.   Figure 1: Example...looking at proteins that are expressed regularly in cells and might be upregulated or downregulated based ...populations. MFI is the average brightness from all cells that are positive for the marker of interest. MFI...

...genetic cargo, typically an expression construct, to cells/organisms. This is often cDNA, shRNA, or something... goal, a promoter should flank this component as well. On both sides of this promoter-gene combo are two...repeat regions in addition to their unique regions as well, though. Viral packaging and entry The genetic cargo...perform very similar functions to infiltrate the host cell and deposit viral cargo. Gene expression While AAV...what will be delivered and expressed in the host cell from the transfer plasmid. Viral packaging and entry...altered tropism of AAV to target different tissue or cell types. AAV also requires some back up from adenovirus...other. Below we will outline the common features as well as the differences within their viral elements. ...

...Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb...Kampmann M, Weissman JS. Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029. Epub 2014 Oct...Can you engineer CRISPR-Cas9 activators that work well enough on any gene that they could be used by people...screening for genes that, when turned on, make melanoma cells resistant to treatment with a BRAF inhibitor. (The...Addgene, enabling systematic investigation of the cellular consequences of repressing or inducing individual...Weissman and his colleagues demonstrated in a paper in Cell that they could typically achieve 90-99% knockdown...straightforward to apply the new library for use in various cell types and in live mice. Therapeutic uses in humans...

...use electro-competent cells instead of the more common chemically-competent cells. You should perform, ...This way you can then cut the plasmid backbone as well as the insert with EcoRI and HindIII and, when you...and verification required for the process to work well, so let’s go over the details step-by-step. 1. ...varies for different enzymes. Many companies now sell fast digest enzymes that can digest large amounts...backbone from the enzymes used to digest them as well as any pieces cut out or off of them. An easy way...undigested controls) are loaded at the top of the gel in wells positioned toward the cathode (- charge). When the... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...

...designed an RNA bait containing three well conserved but not well characterized RNA motifs. This bait identified...proteins that bind an RNA motif of interest in living cells. Challenges with existing RNA-protein interaction...and naturally occurring complexes present in the cell, it can also introduce non-physiological RNA-protein...a short timescale, such as during drug action or cell signaling. Limitations of RaPID Like other BirA*... controls include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA...protein that’s highly expressed in neural progenitor cells. Knock-down of QKI decreased Zika viral RNA levels...2018). RNA–protein interaction detection in living cells. Nature Methods, 15, 207-212. PubMed PMID: 29400715...

...protein of interest in cells and tissues. Although antibody production is well established, it’s also .... RANbodies are expressed in 293T cells and are secreted from cells due to their IgGκ signal peptide sequence... sequence, making them easy to harvest from cell culture media. Their His tags allow for further purification...RANBody While all three reporters stain cultured cells and tissue sections, some reporters are better suited... signal amplification of HRP Staining cultured cells and tissue sections Amplifying weak signals of fluorescent...Labeled anti-HA or -Myc antibody Staining cultured cells and tissue sections When detected with a labeled...Labeled Anti-Chicken IgY antibody Staining cultured cells and tissue sections Great for multicolor staining...

...Many thanks to our guest blogger Gary McDowell! Gary S. McDowell is currently the Executive Director of...This post was contributed by guest blogger, Gary McDowell, executive director of Future of Research. On ...opportunity of the lapsed mandate (or are being compelled at the state level, in the case of some public...expecting a salary increase have had that increase cancelled. Rounding out the remainder, 2% of postdocs who... are continuing with their plans, and 17 have cancelled their plans. 2 of those raising - the University...Illinois and the University of Michigan - initially cancelled their plans, but have since reversed their position... In addition, PIs at some of the institutions cancelling plans are going ahead with raises anyway, including...

...are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is degraded... within cells or tissues- they don’t actually chemically stabilize the RNA molecules (Allewell and Sarma...RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and...before RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues...keeping your samples cold until homogenization and cell lysis. This can be challenging depending on your...developed ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo...is not an issue.  References                 Allewell NM, Sama A (1974) The effect of ammonium sulfate...

...indicator. Archon1 can embed itself into the cell membrane where cell voltage can then be measured. Archon1 ... it. Using a robotic picking approach to select cells with variant characteristics they wanted, the team...2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning podcast segment...through recombination. The 2in1 cloning system is well suited for protein-protein interaction (PPI) studies...offers a way to spatially and temporally control cellular processes in vivo. Many photoactive proteins exist...approach to control protein-protein interactions in cells because the protein binders can be customized for...photo-reversible protein-protein interactions can control subcellular localization processes in vivo. They deposited...

...protocol, we can prep two 96 well plates in just under 3 hours or one 96 well plate in about an hour. Automating...our set up. The cooling block is used to keep our cells from warming up too much and dying.  Also, we added...added a couple of platforms that fit 48 well q- trays allowing us to do up to 96 transformations in one...simple. By clicking a few buttons you can quickly tell the Biomek FxP how much sample to pick up, where...pick up 96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position...be useful if you’re transferring samples from 96 wells plates onto agar q-trays where the samples must ...Have you ever wondered who fills all those vials? Well, it used to be a lab assistant, but with increased...

...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Sajwan S, Mannervik...and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/10.1016/j.cell.2014.09.039 Zhou H, Liu J,...from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation...large and are not expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex ...activator used in other CRISPR activation methods, as well as two other potent transcriptional activators (...activation levels than SAM in some loci across Drosophila cell lines (Sajwan et al., 2019). SPH SPH combines components... Gabrielle Clouse...

...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...allow you to study natural genetic circuits in the cell. Natural and synthetic product production A major...biofuels, drug precursors, etc., microbially or in cell-free systems. These pathways typically involve multiple... be used to assemble multiplexed gRNA systems as well as additional components such as selectable markers...