...toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(...tRNAs are among the most abundant molecules in a cell, this system is clearly also robust enough to ensure...advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing...

... how it works. The findings in a recent issue of Cell demonstrate how PGC-1a1, a transcriptional coactivator... in a commentary about the new work published in Cell Metabolism. But, they note, there is plenty more...JR, Erhardt S, Lindskog M, Ruas JL. doi: 10.1016/j.cell.2014.07.051. PMID: 25259918 Muscle over mind. Moon...

...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins...Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https://doi.org/10.1016/j.cell.2017.03.028 Roth BL (2016...small molecules to elicit a response in a certain cell type. Chemogenetics is similar to optogenetics but...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...to the seven alpha helices that transverse the cellular membrane. The receptor recognizes the appropriate...signaling is initiated when a ligand binds to the extracellular surface of the GPCR. This results in a conformational...

...Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Additional resources... also detect low-frequency cancer mutations from cell-free DNA fragments as well as health-related single...similar types of HPV, HPV16 and HPV18, from human cells, at atomolar levels, within one hour. DETECTR thus...of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https://doi.org/10.1016/j.molcel.2015.10.008...

... brain regions. You can partially control which cells are successfully infected by an AAV by altering ...control where your gene of interest is expressed.  A cell-type specific Cre can provide you with that control...

...mixture of two base editor enzymes in mammalian cells. Read the paper in bioRxiv Find Target-ACE   CRISPR...bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target activity ...

...multiple plasmids in one cell, it might be that you can't stably maintain them in the cell. This blog post features...

...pathways that are frequently implicated in cancer cell proliferation, survival, differentiation, and apoptosis...pathway. “The idea is that if you introduce them into cells one at a time, you could identify those that confer...

... GFP, enabling you to select cells with high GFP expression. These cells have the highest levels of Cas9... on the same plasmid, you’ll make sure that each cell that gets the plasmid contains all of the desired...gRNA, which causes hypopigmentation, thus marking cells that have undergone genomic modification. In this..., et al. “Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.” Scientific...

...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...rapid assessment of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/...

...lack uracil, but contain methionine. (This allows cells carrying nonfunctional as well as functional MET... is available at our Investigations in Molecular Cell Biology Website. Many thanks to our guest blogger...right) has taught courses in biochemistry, molecular cell biology and genetics at Boston College. She is particularly...

... monitoring sensitized emission in living plant cells." (2013). PubMed PMID: 24194740. PubMed Central ...-molecule FRET for imaging biomolecules in live cells (Sustarsic and Kapanidis 2015), which may lead to...understanding biomolecular structure and dynamics in live cells." Current opinion in structural biology 34 (2015...fluorescent proteins available. Fortunately, several excellent reviews have collated data from various papers... 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...

...currently doing this using two approaches: single-cell RNA-sequencing to identify neuronal class and deep...WDDD aims to acquire quantitative data on all the cell divisions of the C. elegans embryo when all essential...Wang et al., 2018).” With the split system, only cells that have both halves of cGAL can drive expression...

...or G->A transitions in a target sequence in live cells. This technique uses catalytically inactive CRISPR...and other means of generating mutations in live cells are all discussed further in this informative post...Clearly we have all of our writers to thank for the excellent content they’ve churned out in the last month....

...allowing for direct comparison of Cre+ and Cre- cells. While Cre-lox recombination is a very useful tool...promoter like CAG, or expressed only in a subset of cells under a more specific promoter (e.g. Rho-Cre is ...promoters. Expression of both N and CCre in the same cell type allows for recombination of LoxP flanked DNA...pNrl-Cre Cre-Myc, Expressed in rod photorecetor cells in the retina Nrl Mammalian Cepko 13962 pBS mPrx1... Okano 153207 pAM-AAV-mSncg-Cre Retinal ganglion cell-specific expression of Cre Mouse Sncg AAV Hu 154018...of loxP plasmids, reporter genes indicate which cells have undergone Cre recombination. These plasmids...37402 Cre Stoplight 2.4 Zsgreen is expressed in cells without Cre, mCherry is expressed with Cre Mammalian...

...small Cas protein, is active in E. coli and human cells Two years ago, Jennifer Doudna’s lab discovered ...demonstrating that CasX is active in both E. coli and human cells. Using the structure of CasX at eight different ...

...tag and small molecule. It does work in mammalian cells, mice, and even yeast, though (we have a kit!). ...and complete degradation of proteins in mammalian cells. Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):E3350...Tags Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...uses a small molecule, a tag, and the endogenous cellular degradation machinery to function. The dTAG is...

... check viral transgene expression in transduced cells whenever possible. This post will describe our workflow...fragments of DNA from the helper plasmids or the cell genome can be packaged inside the vectors during...earlier, it is common to find DNA from the packaging cell genome, bacterial genomes, cloning vectors, and ...

...overexpress YFG in a Drosophila cell line. Knock downs in Drosophila cell lines are extremely effective....person. Animals are diploid except for their germ cells (sets of genomic material are contributed by mother...

... localization of that biomolecule inside living cells. FPs classified by the emission color (emission ...proteins and their applications in imaging living cells and tissues”  Physiol. Rev. 90:1103. Wu et. al. ... nuclear localization, and dynamics” Curr. Opin. Cell. Biol. 23:310. .... Therefore, these can be used as “timers” for cellular processes following their activation. The four...Biosensors: Throwing color lights on molecular and cellular processes” Curr. Protein. Pept. Sci. 9(4):338....

... remember what happened when drug X was added to cell line Y, or the phenotype of a mouse with a specific...comparison of the same experiment from different cell lines/model systems ReFigure allows you to make...

...predict that we’d be able to edit RNA in mammalian cells with CRISPR? I certainly didn’t, but now we can ...Improve HDR Efficiency for CRISPR Editing in Human Cells Day 3: Cpf1: Cpf1: A New Tool for CRISPR Genome ...

...triple colocalization is possible even when staining cells with three IgG1 antibodies. Antibodies were individually...workflow yields similar localization patterns as cells stained for one target (compare nanobodies staining...–rabbit IgG secondary nanobodies. The Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from...

...example, "while it is possible to grow liver stem cells and liver-like mini-organs or organoids, neither...combine bioengineered scaffolds with patient stem cells to generate compatible functional livers.” Why are... optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance...

...mismatched translation rates and potential effects on cell growth, even hosts producing alternative tRNAs may...inclusion bodies that will generally segregate with the cell pellet during purification procedures. Even if you...a means to fine-tune gene expression." Molecular cell 59.2 (2015): 149-161. PubMed PMID: 26186290. PubMed...reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs that recognize different...

...score (KO-score) which represents the proportion of cells that have either a frameshift or 21+ bp indel. This...Following delivery of CRISPR components into target cells, genomic DNA from both edited and unedited (control... score. KO Score - Represents the proportion of cells that have either a frameshift or 21+ bp indel. This...

... . Steps of an IP: 1) Protein extracts from cells or tissues contain a complex mix of proteins seen...common immobilization methods utilizes bacterial cell wall proteins, Protein A and Protein G, which bind...does not. Common negative controls include knockout cell lines or tissues that do not express the protein...

...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen... is primarily a means of selection that targets cells in which homologous recombination has not occurred...ensures high editing efficiency; the only non-edited cells to survive have inactivating mutations in the Cas9...one or more genes in E. coli. and in a mammalian cell line (Qi et al., 2013). Two important features of...the bacteria are unable to resolve them, and the cells die.  This could be applied to diseases where the...

...activator domain (VP64 or p65). Upon expression in the cell, the dCas9-CIB1 fusion binds to the target DNA sequence...activator domain VP64. Using this system in HEK293T cells to induce expression of human IL1RN, the researchers...protein with three red fluorescent tags in U2OS cells. Upon light activation, dCas9 is released within... silencing of transferrin receptor CD71 in HeLa cells.   Comparison of photoactivatable CRISPR strategies...Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light‐Activated Guide RNA. Angew...encoded tools, such as microbial opsins, to control cellular activities using light. In 2015, scientists combined...

...real-time interrogation and monitoring of brain cells and circuits during behavior. Long desired by researchers...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Human...A somatic channelrhodopsin, which enables single cell, single millisecond resolution optogenetics. Cre-dependent...genetically encoded dopamine sensor RdLight1 in mammalian cells. Lin Tian 125708-AAV9 pAAV-hSynapsin1-RdLight1 Expresses...neurons Yulong Li 158757-AAV8 pAAV-CAG-SomaGCaMP6f2 Cell body-targeted GCaMP6f, under CAG promoter: GCaMP6f... Edward Boyden 158757-AAV9 pAAV-CAG-SomaGCaMP6f2 Cell body-targeted GCaMP6f, under CAG promoter: GCaMP6f...

...expanding the genetic code in bacteria and mammalian cells....codons, and tRNA synthetases are introduced into the cell on plasmids and the new amino acid is introduced...Once these plasmids have been introduced in the cells, the non-canonical amino acid can be incorporated...match the growth medium, ncAA concentration, and the cell lines used previously. Remember that the orthogonal... – GFP for E. coli or mCherry-GFP for mammalian cells. You should also express the protein with and without...tRNA synthetase for use in E.coli and Mammalian Cells. Many of the plasmids also contain one or more copies...

... a common thread at the meeting. The new Somatic Cell Genome Editing program, run through the NIH Common...Improve HDR Efficiency for CRISPR Editing in Human Cells Hands-on tips for repair template design from guest...to learn much more about cellular DNA repair pathways. An emphasis on cellular repair post-CRISPR cleavage..., which alter genomic DNA without the need for cellular HDR.   Having had time to digest my notes, since...

... can be used to visualize cells and to quantify targeting of particular cell types. For example, the mDlx...

... life that remained unexplored. Using new single-cell sequencing approaches, the lab was able to eliminate...sample was separated into smaller samples of 5-10 cells each for sequencing, allowing for lower complexity...

...about synthetic promoter AAVs for cell-type expression in retinal cells Resources on Addgene.org Find ...

... can be used to deliver fluorescent proteins to cells as determined by the serotype. You can use these...determine if your AAVs are getting to the right cells using your particular delivery technique and conditions...

...properly. Directly transform chemically competent cells with the digested mixture. There is no need for ...purification before transformation. After transforming cells, you should perform colony PCR with vector specific...

...components or take advantage of existing GFP-lines for cell-specific manipulation. Plasmids Fluorescent Protein...Fluorescent Protein Resources: Empty Backbones FRET Subcellular localization Biosensors Optogenetics Background...

...experiments possible in a wide range of organisms and cell lines. In C. elegans, one of the model organisms... efficiency. Finally, as shown in Drosophila S2 cells (Böttcher et al., 2014), inactivation of non-homologous... community. Developments in mammalian and yeast cells using modified Cas9 proteins to regulate gene expression...homologous recombination donors in cultured Drosophila cells. Nucleic Acids Research 42:e89–e89 . https://doi.org...

...delivered as a pooled library via a lentiviral vector to cell lines that either stably expressed Cas9 or that ...almost all genes involved in the production of a cell surface glycan that’s required for ricin uptake....reveals pathways underlying ricin susceptibility." Cell 152.4 (2013): 909-922. Pubmed PMID: 23394947. Pubmed...

...optogenetics, ICSS can now be done with neuronal cell-type and temporal precision. In ICSS, a test chamber...differential participation in reward circuitry." Cell reports 8.6 (2014): 1857-1869. Pubmed PMID: 25242321...Dynamics of retrieval strategies for remote memories. Cell. 2011;147(3):678-89. Pubmed PMID: 22179551. Pubmed... remote contextual memories. This study is an excellent example in which the authors started with a brain... research. Derek P. Simon received his PhD in Cellular and Molecular Biology from the University of Michigan...

...engineering project at BosLab. Once he optimizes plant cell culture and transgenesis he wants to share his knowledge... in an Open Source Lab: from CRISPR to Cats Stem Cell Models for Disease & Open Science: Interview with...

...these issues by creating immortal antibody-producing cell lines (hybridomas) that secrete antibodies in culture...reproducible production. However, even clonal hybridoma cell lines can produce more than one mAb (Bradbury et...

.... This tag is not suitable for use in apoptotic cells as Caspase3/7 both cleave after the DVPD sequence...fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry, or immunofluorescence...

...regulators with diverse roles in processes such as cell proliferation, differentiation, inflammation, metabolism...

...listed below have the CMV promoter for mammalian cell expression and a T7 promoter for in vitro transcription...

... that converts sucrose into a toxic metabolite. Cells that have undergone a double crossover will not ...contain sacB, allowing them to grow on sucrose. Cells that have only gone through the single cross over... from Harvard. Jake received his MD and a PhD in Cell and Molecular Biology from the University of Pennsylvania...Giacalone et al., 2006). The Chen lab demonstrated excellent counter-selection which enabled very efficient...desired deletion directly into cut vector with excellent efficiency using a commercial isothermal assembly...

...translation of many stop codons and detrimental effects on cell growth. The C321.ΔA strain was genomically recoded...is particularly interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion..."The origins of protein phosphorylation." Nature cell biology4.5 (2002): E127-E130. PubMed PMID: 11988757...commonly cited that approximately one-third of cellular proteins are modified through phosphorylation ...

...EvolvR could also be a tool for lineage tracing cells that do not tolerate double-strand DNA breaks by...researchers to trace the progeny of a single starting cell. Additionally, multiplexing of guides allows EvolvR...

...allow scientists to express a gene of interest in a cell or animal model. AAV plasmids contain several important...Integrity on rAAV8 Production Using the Baculovirus/Sf9 Cells System. Hum Gene Ther Methods, 28(5), 277–289. https...