Retrovirus-mediated gene transfer into canine thyroid using an ex vivo strategy.
We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli beta-galactosidase or Tn7 neomycin-resistance genes. Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418. Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin. To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye DiI, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals. Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7-10 days after transplantation. Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands. Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation. The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation. This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.
Howard Hughes Medical Institute, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
B W
BW
O'Malley
R M
RM
Adams
M L
ML
Sikes
T
T
Sawada
F D
FD
Ledley
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA; D004285:Dogs; D004352:Drug Resistance, Microbial / Q000235:genetics; D008969:Molecular Sequence Data; D009355:Neomycin / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics*; D013961:Thyroid Gland / Q000166:cytology / Q000637:transplantation; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D004247:DNA; D001616:beta-Galactosidase; D009355:Neomycin