Development of a Technique for Introduction of an Expressed Complementary Deoxyribonucleic Acid into Parathyroid Cells by Direct Injection

• Published in: Endocrinology (Philadelphia) Vol. 151; no. 8; pp. 4031 - 4038
• Main Authors: Shiizaki, Kazuhiro, Hatamura, Ikuji, Fukagawa, Masafumi, Nakazawa, Eiko, Saji, Fumie, Watanabe, Yuko, Akizawa, Tadao, Kusano, Eiji
• Format: Journal Article
• Language: English
• Published: United States Endocrine Society 01.08.2010
• Subjects: Adenoviridae - genetics; Animals; Disease Models, Animal; DNA, Complementary - administration & dosage; DNA, Complementary - analysis; DNA, Complementary - genetics; DNA, Complementary - metabolism; Expressed Sequence Tags; Gene Transfer Techniques; Genetic Vectors - administration & dosage; Hyperparathyroidism, Secondary - genetics; Hyperparathyroidism, Secondary - pathology; Injections - methods; Lac Operon; Models, Biological; Osmolar Concentration; Parathyroid Glands - cytology; Parathyroid Glands - metabolism; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Receptors, Calcium-Sensing - administration & dosage; Receptors, Calcium-Sensing - genetics; Receptors, Calcium-Sensing - metabolism; Uremia - genetics; Uremia - pathology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2010-0012

PTH is a major mediator of bone and mineral metabolism. However, physiological and pathological investigations of parathyroid cells (PTCs) have been limited because of the lack of available cell lines and because the organ is too small for detailed studies. Here, we describe a novel method for adenovirus-mediated cDNA transfer into PTCs, and we show the accuracy of the method in a rat model of uremia-induced secondary hyperparathyroidism. Rats underwent a 5/6-nephrectomy and were fed with a high-phosphate diet for 8 wk. The parathyroid glands were surgically exposed and adenoviruses containing LacZ or Ca-sensing receptor (CaSR) were directly injected into the glands under a zoom-stereo microscope. The parathyroid glands were analyzed for infection of adenovirus and immunohistochemically for expression of CaSR. The functional activity of exogenous CaSR in PTCs after this treatment was investigated based on changes of the calcium and PTH curve. A virus concentration of more than 109 plaque-forming units/ml was required for adequate infection of PTCs within 7 d after treatment. Marked increase of CaSR-positive PTCs by 2.39 ± 0.72 times relative to control treatment, and significant colocalization of CaSR overexpression and virus labeling, were observed in glands after gene introduction. The calcium and PTH curve was shifted to the left from the basal position (set point, 1.10 ± 0.09 to 0.76 ± 0.12 mm; P < 0.0001), indicating successful introduction of a functionally active cDNA into the PTCs. This technique may facilitate an elucidation of biological effects through targeting and identification of specific features of PTCs, which may provide the basis for new clinical approaches. An adenovirus-mediated cDNA introduction into parathyroid cells using a direct injection technique might regulate its expression level and biological activity in uremic rat model.