GENE-TRANSFER INTO HUMAN THYROID FOLLICULAR CELLS

The authors established a means of effective gene transfer into human thyroid follicular cells via retroviral-mediated mechanisms. Using specific harvest and culture techniques, we investigated the selection of human thyroid cells in serum-free media. Normal adult human thyroid tissue was obtained after thyroidectomy from fresh specimens sent for frozen section analysis. Follicular cells were harvested and grown in hormonally defined, serum-free media to prevent fibroblast growth with selection for differentiated function assessed by immunohistochemical staining for thyroglobulin. The efficiency of gene transfer into human thyroid cells was compared between the zen-beta-gal and LNL6 retroviral vectors. The zen-beta-gal retrovirus encodes the product beta-galactosidase, and gene expression was demonstrated by histochemical staining in 0.1% to 1% of the cells. An improved efficiency of 2% to 3% transduction was demonstrated using the LNL6 vector which carries the gene for neomycin resistance (NEO-R). Polymerase chain reaction (PCR) identification of the integrated proviral sequence (NEO-R gene) with Southern blot confirmation was used to quantitate LNL6 transductions and compare confluent versus actively dividing cell cultures. Follicular cell. gene therapy has significant potential for treating congenital or acquired diseases of the thyroid as well as disorders of circulating proteins such as diabetes, hypopituitarism, and hemophilia. The ability to culture human follicular cells and perform effective gene transfer is paramount in the eventual realization of thyroid gene therapy.