...type of cells the viral particles infect. This can direct your biosensor to specific tissues or cell (sub...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova...the caffeine of the cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:...blog we will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost...vivo, in tissue preps, in multiple species, and in cell culture models. Some of them have even been expanded... as actinin. When tension is high vs. low in the cell the donor and acceptor of a FRET pair on actinin...many biosensors need to be in specific tissues or cells to monitor their target analyte. This can be difficult...

...Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Find plasmids...are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify... adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable A to I...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . https://doi.org/10.1038...specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:869–874 . https://doi.org/10.1038... modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...

...force to control protein expression in mammalian cells: easy to use biological tools are abundant! In this...our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently induce...tag prior to introduction of the construct into cells. However, if you would like tunable control of an...introduced, they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation. Adapted...be enough, or do you want a permanent control mechanism? Where is the control happening? Is the regulation...need help choosing. Viral expression is another mechanism to integrate your choice cDNA. Both lentivirus...

...domain (Figure 1).  Figure 1: Examples of sites on antibodies used for conjugation. 1) Endogenous ...and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell Biol 89:e115. https://doi.org... like proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et...epitope and transcriptome measurement in single cells. Nat Methods 14:865–868. https://doi.org/10.1038...coupling can be achieved through several different mechanisms. Most take advantage of chemical groups intrinsically...

...Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem Cell 15:27–30 . https://...transcriptome of living cells. Nat Rev Genet 19:770–788 . https://doi.org/10.1038/s41576-018-0059-1 Veres A, Gosis...non-functional allele. For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides,...protein, in order to mitigate the ability of the cell to use an alternative ATG downstream of the annotated...process, and usually involves the need for single cell cloning and subsequent screening for successful ...standard requires not one but two rounds of single cell cloning – as a control, one should revert the edit...passenger variant that came along with the single cell clone (although this is rarely done). When targeting...

...sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses on the enzyme hydropersulfide-... understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: Micrographs...Micrographs of the biosensor expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP...studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute to the ...will explore the connectivity patterns of these cells and the functional contribution to a variety of ...administration and withdrawal to explore how these cells may encode the negative affect associated with drug...library of mutant sensors expressed in a mammalian cell line engineered to be electrically excitable. The...

... attack the cell wall of bacterial cells; however, Mycoplasma species do not possess cell walls. As a ...bacteriophage (viruses that only infect bacterial cells), or a bacterium picking up resistance genes from...conserved, non-negotiable process within the bacterial cell, antibiotic resistance is very likely to develop... to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example, ... communication, and public health.     References 1. O'Neill, J. "Tackling drug-resistant infections globally...another bacterium died for instance). These three mechanisms of acquired drug resistance are referred to as...competition drives microbes to develop survival mechanisms. Some microbes even make their own antibiotic...

...different types of human cells, including T cells and hematopoietic stem and progenitor cells. Now with the DAP...such host-defense mechanisms to enable multiplex gene editing in a variety of cells and organisms. However... over 50% averaging editing efficiency in human cells. MBE with DAP array also exhibited reduced Cas9-... editing, and nuclease editing, in virtually any cell type or organisms of interest with only minimal ... genes with a single viral vector.   Fig. 1: Schematic of multiplex gene editing with DAP array... fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. They... To do so, they harnessed the tRNA processing mechanism, which is conserved in all living organisms and...

...adhesions in living cells in several different contexts.   Figure 5: Left: Mechanism of the PILATeS...Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates mechanical tension, and the...and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want...are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1]...biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https://doi.org/10.1038/s41467-024-52871-9...disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody collection grows...useful addition to the toolkit for visualizing cellular processes in C. elegans: mScarlet3 for applications...

...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...energy source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins...

...References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science...work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase-9 secretion...both Cas9 and your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer...knock-out thousands of genes in mixed population of cells as part of pooled library screens (1,2). The human...pooled libraries targeting both mouse and human cells are avaliable for distribution. To learn more about...Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit our Lentiviral...

...prevent cell death. One of the cool things about amp is that it's exclusively toxic to dividing cells. The... continuous cellular processes (e.g., protein synthesis), while amp only affects the cell wall synthesis...Check icon" %} Spectinomycin Excellent No Just bacteria Not SHuffle cells {% icon icon_set="fontawesome...icon" %} Zeocin Excellent No Bacteria, plants, and eukaryotes Not Tn5-containing cells {% icon icon_set..., penicillin, and is a beta-lactam that inhibits cell wall synthesis. The AmpR (amp resistance) gene produces...synthesis step during cell division. This is nifty because the recovery period after transformation (the dreaded...at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase prior to...

...cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks...its oxidized derivatives. Cell 152, 5 P1146-1159. https://doi.org/10.1016/j.cell.2013.02.004.  Xu X, Tao...targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc.2016.9.   ...methylate sites blocked by dCas9. Within a few rounds of cell division, the original methylated strand is diluted...dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can...can be achieved by fluorescence-activated cell sorting, and stable gRNA expression can be achieved using...TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers. Nat...

...trials in 2021. Figure 1: Schematic of the human MECP2 locus and MECP2 protein, showing mutations ...fine-tuning dosage so that enough wild-type MECP2 is made inside the cells, but this is exciting progress in tackling... also find links to cell line repositories including patient-derived iPSCs, MECP2 animal models, tissue...Domain in MeCP2 Determines the Clinical Course of Rett Syndrome and Related Disorders. Cell 152:984–996...CRISPR/Cas9-Mediated MECP2 Modifications in Human-Induced Pluripotent Stem Cells. Front Genet 10: . https...deliver the precise amount of protein to each brain cell to restore physiological levels. Much research is...repair a gene in a living animal across multiple cell types using a mouse model for a human neurological...

...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material... directly inhibit the ability of a cell to undergo programmed cell death while simultaneously stimulating...eliminate every cancerous cell. Even so, to pose the greatest threat, cancerous cells still need to successfully...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division. This...proteins that, when expressed, normally encourage cell survival, growth and proliferation, while tumor ... suppressors perform the opposite task, keeping cells from dividing out of control. One may compare the...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements...stimulate AraC binding to I1 and I2 sites. Supplementing cell growth media with glucose decreases cAMP and represses... Because transcription machinery differs between cell types or organisms, promoters are similarly variable...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique...there is very little crossover. The induction mechanism must also be compatible with your experimental...

... of cell types in the nervous system. While certain cell types, such as principal excitatory cells and...neuronal cell types involved in neurodegenerative diseases, the Cole Lab selected promoters for cell types...with edited “pools” of cells, eliminating the time-consuming step of single-cell isolation. Agudelo et ...gene. Koo M, et al. Cell Systems. 2016. PubMed PMID: 28189581 Peters J, et al. Cell. 2016. PubMed PMID:...generation Deriving iPSCs (induced pluripotent stem cells) through the expression of transcription factors...the established model for reprogramming somatic cells. A common method for generating iPSCs to express...work are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 Targeting...

...at bioRxiv. AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral...of the packaging cells (Vandenberghe, 2010). Rather than being released into the cell culture supernatant... particles associate with the cell membrane and are taken into the cell via endocytosis. In our study,...AAV2 viral particles by our packaging cells by treating the cells with the endocytosis inhibitor sucrose...target cells is blocked following sucrose treatment. Moreover, several reports have shown that cells treated...untreated and sucrose treated cells. Although we were unable to deduce the mechanism responsible we still believe... yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype specific...

...labeling T cells, B cells, and antigen presenting cells, scientists have been able to decypher cellular dynamics...tracked by cell sorter giving immunologists the ability to count cells and statistically analyze cell movements...fast T cells could jump from one antigen presenting cell to another. Indeed, we now know that T cells are...separates the cells mechanically in a vibrating nozzle, imparting a positive or negative charge to cells that ...2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et al...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during.... a basal progenitor cell migrating to the epithelium). At this stage, only cells from the immune system...

...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610... and immunoglobulin heavy chain binding protein. Cell 46:291–300. https://doi.org/10.1016/0092-8674(86...signal prevents secretion of luminal ER proteins. Cell 48:899–907. https://doi.org/10.1016/0092-8674(87...