Advances and enabling technologies for phase-specific cell cycle synchronisation

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Pritam Bordhan ◽  
Sajad Razavi Bazaz ◽  
Dayong Jin ◽  
Majid Ebrahimi Warkiani
Keyword(s):  
Cell Cycle ◽  
Cell Cultures ◽  
Gene Editing ◽  
Cell Populations ◽  
Specific Cell ◽  
Enabling Technologies ◽  
Targeted Gene Editing

Cell cycle synchronisation is the process of isolating cell populations at specific phases of the cell cycle from heterogeneous, asynchronous cell cultures. The process has important implications in targeted gene-editing...

scholarly journals Loss of Melanopsin (OPN4) Leads to a Faster Cell Cycle Progression and Growth in Murine Melanocytes

2021 ◽  
Vol 43 (3) ◽  
pp. 1436-1450
Author(s):  
Leonardo Vinícius Monteiro de Assis ◽  
Maria Nathália Moraes ◽  
Davi Mendes ◽  
Matheus Molina Silva ◽  
Carlos Frederico Martins Menck ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Population Analysis ◽  
Cell Populations ◽  
Specific Cell ◽  
Significant Modification ◽  
M Cell ◽  
Cycle Progression ◽  
Gene And Protein Expression ◽  
Independent Functions

Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0–G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.

scholarly journals Multiparametric Cell Cycle Analysis by Automated Microscopy

2006 ◽  
Vol 11 (6) ◽  
pp. 586-598 ◽  
Author(s):  
Fabio Gasparri ◽  
Paolo Cappella ◽  
Arturo Galvani
Keyword(s):  
Cell Cycle ◽  
Cell Cultures ◽  
Dna Content ◽  
Cyclin B1 ◽  
Cell Cycle Analysis ◽  
Brdu Incorporation ◽  
Specific Cell ◽  
Drug Mechanism ◽  
Automated Microscopy ◽  
Cellular Dna

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics’s ArrayScan™ reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.

scholarly journals Data Driven Cell Cycle Model to Quantify the Efficacy of Cancer Therapeutics Targeting Specific Cell-Cycle Phases From Flow Cytometry Results

2021 ◽  
Vol 1 ◽  
Author(s):  
David W. James ◽  
Andrew Filby ◽  
M. Rowan Brown ◽  
Huw D. Summers ◽  
Lewis W. Francis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Phase Transitions ◽  
Mechanism Of Action ◽  
Cell Cycle Phase ◽  
Data Driven ◽  
Cycle Phase ◽  
Cell Populations ◽  
Specific Cell ◽  
Cell Cycle Phases

Many chemotherapeutic drugs target cell processes in specific cell cycle phases. Determining the specific phases targeted is key to understanding drug mechanism of action and efficacy against specific cancer types. Flow cytometry experiments, combined with cell cycle phase and division round specific staining, can be used to quantify the current cell cycle phase and number of mitotic events of each cell within a population. However, quantification of cell interphase times and the efficacy of cytotoxic drugs targeting specific cell cycle phases cannot be determined directly. We present a data driven computational cell population model for interpreting experimental results, where in-silico populations are initialized to match observable results from experimental populations. A two-stage approach is used to determine the efficacy of cytotoxic drugs in blocking cell-cycle phase transitions. In the first stage, our model is fitted to experimental multi-parameter flow cytometry results from untreated cell populations to identify parameters defining probability density functions for phase transitions. In the second stage, we introduce a blocking routine to the model which blocks a percentage of attempted transitions between cell-cycle phases due to therapeutic treatment. The resulting model closely matches the percentage of cells from experiment in each cell-cycle phase and division round. From untreated cell populations, interphase and intermitotic times can be inferred. We then identify the specific cell-cycle phases that cytotoxic compounds target and quantify the percentages of cell transitions that are blocked compared with the untreated population, which will lead to improved understanding of drug efficacy and mechanism of action.

scholarly journals Chronic, cortex-wide imaging of specific cell populations during behavior

2021 ◽  
Author(s):  
Joao Couto ◽  
Simon Musall ◽  
Xiaonan R. Sun ◽  
Anup Khanal ◽  
Steven Gluf ◽  
...  
Keyword(s):  
Cell Populations ◽  
Specific Cell

scholarly journals Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Gene Therapy ◽  
2021 ◽  
Author(s):  
Jonathan O’Keeffe Ahern ◽  
Irene Lara-Sáez ◽  
Dezhong Zhou ◽  
Rodolfo Murillas ◽  
Jose Bonafont ◽  
...  
Keyword(s):  
Delivery System ◽  
Gene Editing ◽  
Transfection Efficiency ◽  
Attractive Alternative ◽  
Safe Delivery ◽  
Guide Rna ◽  
Recessive Dystrophic Epidermolysis Bullosa ◽  
Targeted Gene Editing ◽  
Genomic Editing ◽  
Polymeric Vectors

AbstractRecent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

scholarly journals NAC, Tiron and Trolox Impair Survival of Cell Cultures Containing Glioblastoma Tumorigenic Initiating Cells by Inhibition of Cell Cycle Progression

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e90085 ◽  
Author(s):  
Massimiliano Monticone ◽  
Razieh Taherian ◽  
Sara Stigliani ◽  
Elisa Carra ◽  
Stefano Monteghirfo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cultures ◽  
Cell Cycle Progression ◽  
Cycle Progression

scholarly journals Cell cycle analysis of primary sponge cell cultures

2011 ◽  
Vol 47 (4) ◽  
pp. 302-311 ◽  
Author(s):  
Klaske J. Schippers ◽  
Dirk E. Martens ◽  
Shirley A. Pomponi ◽  
René H. Wijffels
Keyword(s):  
Cell Cycle ◽  
Cell Cultures ◽  
Cell Cycle Analysis ◽  
Sponge Cell

scholarly journals High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.

1997 ◽  
Vol 17 (9) ◽  
pp. 5588-5597 ◽  
Author(s):  
A Sewing ◽  
B Wiseman ◽  
A C Lloyd ◽  
H Land
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cell Cycle Arrest ◽  
Expression Patterns ◽  
Cellular Responses ◽  
Specific Cell ◽  
Cyclin D ◽  
Inhibition Of Proliferation ◽  
Cycle Arrest ◽  
Signal Specificity

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.

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