Hsieh Fj

Molecular Analysis and Characterization of Zebrafish Keratocan (zKera) Gene

Corneal small leucine-rich proteoglycans play a pivotal role in maintaining corneal transparency and function. In this study, we isolated and characterized the zebrafish (Danio rerio) keratocan (zKera) gene. The human keratocan sequence was used to search zebrafish homologues. The zKera full-length genomic DNA and cDNA were generated via PCR of zebrafish genomic DNA and reverse transcription-PCR of total zebrafish eye RNA, respectively. The zKera spanning 3.5 kilobase pairs consists of two exons and one intron and a TATA-less promoter. The zKera encodes 341 amino acids with 59% identity to its human counterpart and 57% identity to that of mouse keratocan. Like mouse and chick keratocan, zKera mRNA is selectively expressed in the adult cornea; however, during embryonic development, zKera mRNA is expressed in both the brain and the cornea. Interestingly, it is expressed mainly in corneal epithelium but also in the stroma. A pseudogene was proved by introducing a zKera promoter-driven enhanced green fluorescence protein reporter gene into fertilized zebrafish eggs. Using morpholino-antisense against zKera to knock down zKera resulted in a lethal phenotype due to massive caspase-dependent apoptosis, which was noted by a significant increase of active caspase-3 and caspase-8 in the developing forebrain area, including the eyes. This is different from mouse, for which keratocan-deficient mice are viable. Taken together, our data indicate that mammalian keratocan is conserved in zebrafish in terms of gene structure, expression pattern, and promoter function.

Three-dimensional ultrasonography in genetic screening and counselling

ABSTRACTThree-dimensional ultrasound was introduced in clinical use more than a decade ago. Early attempts in obstetrics were directed at surface rendering of the fetal anatomy, mostly of the face, to detect or exclude fetal anomalies. Recently, several papers have discussed fetal volumetry calculated by three-dimensional ultrasound, which has helped greatly to construct the normal ranges for fetal organ volumes, and which improves fetal weight prediction. According to recent literature, more and more fetal anomalies are diagnosed based only on the findings of three-dimensional ultrasound. However, the role of three-dimensional ultrasound in genetic screening and counselling is still undetermined. In this review, we try to address the capability of three-dimensional ultrasound in a number of anatomic areas, to catalog possible markers in the second-trimester genetic sonogram, and finally to introduce the merits of three-dimensional ultrasound in genetic counselling.