PubMed · 15105580
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V Girish, A Vijayalakshmi. Affordable image analysis using NIH Image/ImageJ.. https://pubmed.ncbi.nlm.nih.gov/15105580/
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Platforms that allow parallel, quantitative analysis of single cells will be integral to realizing the potential of postgenomic biology. In stem cell biology, the study of clonal stem cells in multiwell formats is currently both inefficient and time-consuming. Thus, to investigate low-frequency events of interest, large sample sizes must be interrogated. We report a simple, versatile, and efficient micropatterned arraying system conducive to the culture and dynamic monitoring of stem cell proliferation. This platform enables: 1) parallel, automated, long-term ( approximately days to weeks), live-cell microscopy of single cells in culture; 2) tracking of individual cell fates over time (proliferation, apoptosis); and 3) correlation of differentiated progeny with founder clones. To achieve these goals, we used microfabrication techniques to create an array of approximately 10,000 microwells on a glass coverslip. The dimensions of the wells are tunable, ranging from 20 to >500 microm in diameter and 10-500 microm in height. The microarray can be coated with adhesive proteins and is integrated into a culture chamber that permits rapid (approximately min), addressable monitoring of each well using a standard programmable microscope stage. All cells share the same media (including paracrine survival signals), as opposed to cells in multiwell formats. The incorporation of a coverslip as a substrate also renders the platform compatible with conventional, high-magnification light and fluorescent microscopy. We validated this approach by analyzing the proliferation dynamics of a heterogeneous adult rat neural stem cell population. Using this platform, one can further interrogate the response of distinct stem cell subpopulations to microenvironmental cues (mitogens, cell-cell interactions, and cell-extracellular matrix interactions) that govern their behavior. In the future, the platform may also be adapted for the study of other cell types by tailoring the surface coatings, microwell dimensions, and culture environment, thereby enabling parallel investigation of many distinct cellular responses.
Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway.
Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.