Circadian modulation of hepatic transcriptome in transgenic rats expressing human growth hormone.
ABSTRACT The secretory profile of growth hormone (GH) is sexually dimorphic in rats. In male transgenic (TG) rats expressing human GH (hGH) that we generated, the circulating levels of both hGH and endogenous GH are flattened with no male-type pulsatility. To elucidate the regulatory role of episodic GH profile on the liver, the hepatic transcriptome of male TG rats at the middle of the light and dark phases was characterized by genome-wide analyses as compared with that of male wild-type (WT) rats. Transcripts commonly up- or down-regulated regardless of the lighting conditions in TG rats were mainly enriched in the metabolism of xenobiotics. In TG rats, the gene expression profile was functionally feminized, verifying that the sexually dimorphic profile of GH rather than genetic sexuality is a stronger sex-determining factor on the hepatic transcriptome. The common transcripts which fluctuated during the day in both TG and WT rats were enriched in circadian rhythm signaling, and physiological rhythmicity was considered to be finely interconnected with liver metabolism via sexually dimorphic GH secretion. In contrast, some genes were differentially regulated in TG rats at only one of two time points measured, and others were fluctuated daily in only one genotype. In particular, some genes involved in the GH signaling pathway were included, suggesting the signal transduction is circadian-modulated depending upon the GH profile. Our transcriptome analyses clarified the regulatory role of episodic GH profile on the liver and strengthen the functional link between sexually dimorphic GH secretion, liver metabolism, and its circadian regulation.
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ABSTRACT: A chimeric gene comprising murine whey acidic protein (mWAP) and human growth hormone (hGH) was used to produce transgenic rats that express hGH and secrete it into the blood. Two lines of transgenic rats carrying the mWAP/hGH construct were established: Line 1 was characterized by relatively high levels of serum hGH, and Line 2 had relatively low levels. The secretory profiles of rat GH (rGH) as well as hGH, the transgene product, were obtained in transgenic males and females of Line 2; both hGH and rGH serum levels were flattened with no episodic fluctuations, and the overall mean concentration of rGH was significantly lower than in normal littermates. Although the animals of Line 1 showed an accelerated increase in growth rate, those of Line 2 did not. Nevertheless, the onset of puberty in females, as assessed by vaginal opening and occurrence of first ovulation, advanced by 7-8 days in both Lines of animals. Accordingly, the body weight at puberty of Line 2 transgenic females was much lower than that of normal littermates, indicating that continuous hGH expression could induce precocious puberty without enhancing the growth rate.Endocrine Journal 11/1994; 41(5):523-9. · 2.23 Impact Factor
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ABSTRACT: A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.Genome Research 11/1996; 6(10):995-1001. · 14.40 Impact Factor
Article: Real time quantitative PCR.[show abstract] [hide abstract]
ABSTRACT: We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over contamination and resulting in much faster and higher throughput assays. The real-time PCR method has a very large dynamic range of starting target molecule determination (at least five orders of magnitude). Real-time quantitative PCR is extremely accurate and less labor-intensive than current quantitative PCR methods.Genome Research 11/1996; 6(10):986-94. · 14.40 Impact Factor