Jian Sheng Sun | PubMed Health

PubMed2005

Smart 3D-FISH: automation of distance analysis in nuclei of interphase cells by image processing.

BACKGROUND: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved three-dimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. METHODS: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. RESULTS: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4',6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of spot loci in the nucleus and inter-spot distance. Results from a pilot experiment using Smart 3D-FISH on ENL, MLL, and AF4 genes in two lymphoblastic cell lines were satisfactory and consistent with data published in the literature. CONCLUSION: Smart 3D-FISH should greatly facilitate image processing and analysis of 3D-FISH images by providing a useful tool to overcome the laborious task of image segmentation based on user-defined parameters and decrease subjectivity in data analysis. It is available as a set of plugins for ImageJ software.

Algorithms↗

PubMed2003

Tyrosinase gene correction using fluorescent oligonucleotides.

Gene therapy and production of mutated cell lines or animal models should be improved significantly once efficient controlled gene targeting strategies are developed. We used short single-stranded oligodeoxynucleotides (ODN), in some cases coupled to the fluorescent dye fluorescein isothiocyanate (FITC), to correct an endogenic natural point mutation in melanocytes in culture. The addition of the FITC molecule to the 5' extremity of the ODN did not interfere with the efficiency of the reversion of the mutation and did not have any deleterious side-effects. The use of fluorescent ODN could lead to great improvement in the technique. In particular, it may facilitate sorting of the transfected cells in the treated population, and thereby significantly increase the percentage of corrected cells.

Animals↗

PubMed2002

Binding properties of oligonucleotides containing a modified 2'-deoxyuridine with a thymine ended linker to pair with 2'-deoxyadenosine.

Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2'-deoxyuridine harbouring at C-5 a thymine ended linker, were found to undergo base pairing with the opposite 2'-deoxyadenosine. However, the corresponding duplexes are significantly destabilised as compared to the fully natural ones.

Binding Sites↗