327 CHARACTERIZATION OF FETAL AND ADULT FIBROBLASTS FROM NELORE BOVINE FOR NUCLEAR TRANSFER

Abstract

Cloning by nuclear transfer is a technology that has provided major advances in reproductive sciences. It is an efficient method: (1) to produce transgenic embryos, fetuses, and animals; (2) to multiply genetically superior adult animals; and (3) to generate proteins, tissues, and organs for xenotransplants of human interest. Basic studies on nuclear transfer have contributed to understanding how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies. The aim of this study was to establish a stable lineage of Nelore adult and fetal fibroblasts for embryo reconstruction. Cultures were characterized by morphological aspects, and by immunocytochemical and ultrastructural analysis. Cultures of fetal and adult fibroblasts at passage 2 stained positively for cytokeratin, a typical protein from the intermediate filament of epithelial cells, in some cells but staining was not found in subsequent passages. A dense and organized network occurred in adult and fetal fibroblast cultures presenting positive immunostainig for vimentin in pass 5, 10, and 15 subcultures, but did not stain for cytokeratin (negative control). The results demonstrated that fibroblasts continued expressing vimentin and maintained their morphological aspects in culture. The ultrastructural analysis showed the presence of organelles involved in protein synthesis including dilated rough endoplasmatic reticulum, Golgi cisternae, and polyribosomes. Elongate mitochondria were distributed around the Golgi complex, suggesting high metabolic activity. These results demonstrated that adult and fetal fibroblasts continued to develop biological activities and maintained specific characteristics under these culture conditions. In conclusion, adults and fetal fibroblasts can be successfully used as donor cells for nuclear transfer.

This work was supported by FAPESP 01/11931-8 and 01/13944-0.