Quantification of growth hormone receptor extra- and intracellular domain gene expression in chicken liver by quantitative competitive RT-PCR

The very sensitive competitive reverse transcription-polymerase chain react ion (RT-PCR) was used to investigate the expression of the extracellular (G HRe) and intracellular (GHRi) parts of the growth hormone receptor (GHR) in the liver tissue of chickens. Two competitors (internal standards), pGHRi MUT and pGHRe MUT, specific to the GHRi and GHRe genes, respectively, were constructed by site-specific mutagenesis. The internal standards defined PC R products of 394 bp for the pGHRi MUT and 330 bp for the GHRe MUT. These w ere used as competitors to the wild-type GHRi or GHRe which defined PCR pro ducts of 382 and 328 bp, respectively. Coamplification, under standardized conditions, of the native RNA in competition with serial dilutions of the m utant RNA in the same PCR reaction followed by enzymatic digestion produced the expected sizes of internal standard cDNA and predicted target cDNA. Ex pression Levels of GHRe and GHRi were determined from standard curves gener ated. The method was sensitive enough to detect expressions down to picogra m levels. Applying this method, the effect of GH and T-3 injection on GHRe and GHRi mRNA expression was determined in the liver of adult female Hisex birds and 1-day-old normal and dwarf chickens. Intravenous GH injection (25 mug/kg body weight) increased plasma levels of GH in Hisex birds after 10 min but rapidly decreased at 60 min followed by an increase in T-3. GH inje ction significantly increased the expression of the GHRe 60 min after injec tion but not at 10 min, when the CH level in plasma was high. In the liver of saline-treated dwarf (dw) and non-dwarf (Dw) chicks, the level of expres sion of GHRe was similar in both strains despite disparate levels of basal GH and T-3. However, the level of GHRi was higher in Dw than in dw chicks. Although GH levels increased in both strains after intravenous GH injection (250 mug/kg body wt), the expression of GHRe in both strains was unaffecte d. However, the mRNA for the GHRi was significantly depressed by injection in the Dw but unaffected in dw chicks. Intravenous injection of T-3 (0.5 an d 5 mug/kg body wt) increased plasma levels in both strains but caused depr ession of GHRi in Dw but not in dw chicks. T-3 injections had no effect on GHRe in either Dw or dw chicks. It is concluded that the expression of the GHRe in adult chickens is GH regulated either directly or indirectly. In co ntrast, in 1-day-old chicks, GH or T-3 had no effects on the GHRe but regul ated the expression of GHRi in Dw chicks, whereas in dwarf chicks both had no effect on GHRe or GHRi expression. It is postulated that GHRe and GHRi g ene expression may be regulated by different agonists/antagonists in differ ent strains and depending on the age of the chicken. (C) 2001 Academic Pres s.