Isolation of carp cDNA clones, representing developmentally-regulated genes, using a subtractive-hybridization strategy

Abstract

A subtractive-hybridization technique, combined with differential screenings and subsequent whole mount in situ hybridization (ISH) reactions, was used to isolate novel cDNA clones representing developmentally-regulated genes of carp. Small-scale differential screenings of an oocyte and a segmentation-stage cDNA library using oocyte-specific and segmentation stage-specific enriched probes, yielded 75 positive clones. ISH screening showed that 65% (15) of the oocyte-stage clones and 50% (26) of the segmentation-stage clones were indeed stage-specific. Partial sequence analysis suggests that approximately 65% of the 41 stage-specific clones represent novel genes. In addition, an Otxl clone was isolated. Two novel clones and the Otxl clone are of special interest for developmental studies. The clones represent genes that are locally expressed during embryonic development. The expression patterns of Otxl and one of the novel clones suggest functions in specification of the anterior-posterior axis. The three clones provide molecular markers for the study of gastrulation and the patterning of the a-p axis in teleosts.

Use of serial analysis of gene expression to generate kidney expression libraries

“…Gene expression profiles can be generated and compared by multiple techniques. Subtractive hybridization (33), subtraction libraries (14), and differential display (2,5) are semiquantitative methods, which may not detect small, pathophysiologically important variations in gene expression. In contrast, DNA microarrays quantitatively assay expression levels of thousands of genes, using discrete DNA sequences robotically imprinted on glass microscope slides (9), but require technology and instrumentation not available to most investigators.…”