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 spe cific harvest and culture techniques, we investigated the selection of human thyroid cells in serum-free media. Normal adult human thyroid t issue was obtained after thyroidectomy from fresh specimens sent for f rozen section analysis. Follicular cells were harvested and grown in h ormonally 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 retrovir al vectors. The zen-beta-gal retrovirus encodes the product beta-galac tosidase, and gene expression was demonstrated by histochemical staini ng in 0.1% to 1% of the cells. An improved efficiency of 2% to 3% tran sduction was demonstrated using the LNL6 vector which carries the gene for neomycin resistance (NEO-R). Polymerase chain reaction (PCR) iden tification of the integrated proviral sequence (NEO-R gene) with South ern blot confirmation was used to quantitate LNL6 transductions and co mpare confluent versus actively dividing cell cultures. Follicular cel l. gene therapy has significant potential for treating congenital or a cquired diseases of the thyroid as well as disorders of circulating pr oteins 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.