Gene expression and mRNA editing of serotonin receptor 2C in brains of HPRT gene knock-out mice,an animal model of Lesch-Nyhan disease

Lesch-Nyhan disease (LND), a genetic disorder associated with motor and psychiatric disturbance and self-injurious behaviour (SIB) is caused by a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). The connection between enzyme deficiency and neurological involvement is still unclear. Evidence exists for a role of basal ganglia dysfunction with decreased dopamine and excess serotonin striatal content. In this study, we investigate the role of serotonin receptor 2C (HTR2C) in the brains of HPRT gene knock-out mice, a model of LND. HTR2C expression is analyzed by real-time polymerase chain reaction (PCR) using SYBR-green detection methods. The percentage of edited HTR2C mRNA was determined by direct sequencing of amplification products of the region containing the editing sites. We found a 55% increase in the expression of HTR2C gene but no significant difference in mRNA editing levels between knock-out and control mice. The above alteration found in HPRT-deficient mice is similar to those found in other animal models used to study aggressive and self-injurious behaviour.     

Changes in estrogen receptor-alpha mRNA in the mouse cortex during development

Estrogen plays a critical role in brain development and is responsible for generating sex differences in cognition and emotion. Studies in rodent models have shown high levels of estrogen binding in non-reproductive areas of the brain during development, including the cortex and hippocampus, yet binding is diminished in the same areas of the adult brain. These binding studies demonstrated that estrogen receptors decline in the cortex during development but did not identify which of the two estrogen receptors was present. In the current study, we examined the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the mouse cortex during the first month of life. Messenger RNA was isolated from cortical tissue taken from C57BL/6 mice on postnatal day (PND) 1, 4, 10, 18 and 25 and expression levels were determined by real-time PCR. ERalpha mRNA expression in the mouse cortex at PND 25 was significantly reduced as compared to PND 1 (p<0.01). ERbeta mRNA expression at PND 25 was significantly increased as compared to PND 1 (p<0.05). Although the increase in ERbeta mRNA was statistically significant, the ERbeta levels were extremely low in the isocortex compared to ERalpha mRNA levels, suggesting that ERalpha may play a more critical role in the developmental decrease of estradiol binding than ERbeta. Additionally, we measured ERalpha mRNA expression in organotypic explant cultures of cortex taken from PND 3 mice. Explants were maintained in vitro for 3 weeks. mRNA was isolated at several time points and ERalpha and ERbeta mRNA was measured by real-time RT-PCR. ERalpha and ERbeta mRNA levels reflected a similar pattern in vitro and in vivo, suggesting that signals outside the cortex are not needed for this developmental change. This study lays the groundwork for an understanding of the mechanisms of the developmental regulation of ERalpha mRNA.     

The use of real-time PCR analysis in a gene expression study of Alzheimer's disease post-mortem brains

The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.     

Gene expression profiling in Brodmann's area 46 from subjects with schizophrenia

OBJECTIVE: To identify altered gene expression in the dorsolateral prefrontal cortex obtained after death from subjects with schizophrenia. METHOD: Restriction fragment differential display (RFDD) was used to measure levels of mRNA in Brodmann area (BA) 46 from schizophrenia and control subjects. Findings on specific mRNA identified with RFDD were further investigated using real-time polymerase chain reaction (real-time PCR), PCR and western blotting. RESULTS: Levels of mRNA for 63 of approximately 12,500 genes differed in BA 46 in schizophrenia. Subsequent real-time PCR has shown that mRNA for muscleblind protein 1 (MBNL1) and protocadherin 17 (PCDH17) are increased in BA 46 from subjects with schizophrenia of short, but not long, duration. Altered levels of mRNA for neither gene were present in BA 9 from subjects with schizophrenia or in either cortical area from subjects with bipolar 1 disorder. By contrast, both RFDD and real-time PCR failed to show altered expression of the schizophrenia candidate gene disrupted in schizophrenia 1 (DISC1) BA46 from any diagnostic cohort. CONCLUSION: The present study has identified genes that are differentially expressed in BA 46 in schizophrenia. Initial studies have shown that there is a need for a careful validation of genes shown to be affected in schizophrenia using high-throughput technologies. In addition the present study has shown that gene expression may vary considerably depending on the duration of schizophrenia. This raises the hypothesis that changing gene expression may be underlying the change in symptom profile that occurs with disease progression in some subjects with schizophrenia.     

肝豆状核变性基因在皮肤成纤维细胞株转染后的表达

目的利用逆转录聚合酶链反应(RT-PCR)法在核酸水平检测并证实肝豆状核变性(Wilson病,WD)基因可以在Me32aT22细胞株中得到有效表达,为以后更深入的研究奠定基础。方法采用脂质体转染法将WDcDNA重组真核表达载体pRc/CMV-WD导入无血清的Me32aT22/2L细胞株,RT-PCR在核酸水平检测WD基因在Me32aT22/2L细胞株的表达。结果在转染的Me32aT22/2L细胞株中可检测到WDcDNA表达,而在未转染的Me32aT22/2L细胞中RT-PCR检测结果呈阴性。结论WD基因转染至Me32aT22/2L细胞株后能得到有效表达。     

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.     

Quantitation of nicotinic acetylcholine receptor subunits alpha 4 and beta 2 messenger RNA in postmortem human brain using a non-radioactive RT-PCR and CCD imaging system

We present a simple and rapid procedure for quantifying mRNA in the brain after RT-PCR, in which the intensity of the ethidium bromide luminescence of PCR products is measured directly from electrophoretic gels by a highly sensitive CCD camera combined with an image analyzing computer system (Gel Doc 1000 system). The CCD camera allows the mRNA in the ethidium bromide-stained PCR-amplified bands to be quantified in a broad exponential range of PCR cycles. The proposed protocol enables standard curves to be constructed to examine the relationship between the number of reaction cycles and amplified log intensity and between the amount of sample RNA for RT-PCR and amplified intensity. The method was applied to nicotinic acetylcholine receptor (nAChR) subunits alpha 4 and beta 2 mRNA in postmortem human putamen in the present study, but is also applicable to mRNAs of other receptors and neurotransmitter precursor peptides.     

先天性人巨细胞病毒感染胎鼠大脑皮质钙调素基因mRNA的研究

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Behavioral effects of elevated expression of human equilibrative nucleoside transporter 1 in mice

Adenosine concentrations are regulated by purinergic enzymes and nucleoside transporters. Transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1) have been generated (Parkinson et al., 2009 [7]). The present study tested the hypothesis that mice homozygous and heterozygous for the transgene exhibit differences in hENT1 mRNA and protein expression, and in behavioral responses to caffeine and ethanol, two drugs with adenosine-dependent actions. Real time polymerase chain reaction (PCR) was used to identify mice heterozygous and homozygous for the transgene. Gene expression, determined by real time PCR of cDNA reverse transcribed from cerebral cortex RNA, was 3.8-fold greater in homozygous mice. Protein abundance, determined by radioligand binding assays using 0.14 nM [³H]S-(4-nitrobenzyl)-6-thioinosine ([³H]NBTI), was up to 84% greater in cortex synaptosome membranes from homozygous than from heterozygous mice. In western blots with an antibody specific for hENT1, a protein of approximately 40 kDa was strongly labelled in cortex samples from homozygous mice, weakly labelled in samples from heterozygous mice and absent from samples from wild type mice. In behavioral assays, transgenic mice showed a greater response to ethanol and a reduced response to caffeine than wild type littermates; however, no significant differences between heterozygous and homozygous mice were detected. These data indicate that the difference in ENT1 function between wild type and heterozygous mice was greater than that between heterozygous and homozygous mice. Therefore, either heterozygous or homozygous hENT1 transgenic mice can be used in studies of ENT1 regulation of adenosine levels and adenosine dependent behaviors.