Core--Real-time quantitative PCR facility

实时定量聚合酶链式反应核心的目的是帮助研究人员为代谢疾病基因治疗项目评估基因转移效率和目标细胞中的转基因表达。正如目前的基因治疗临床试验(亨特综合征的淋巴细胞基因治疗)初步证明的那样，这项技术满足了非常重要的科学需求，即准确地定量某个基因的存在，并用高灵敏度和重复性的逆转录-聚合酶链式反应(RT-PCR)来测量其表达。该核心将帮助研究人员设计分析系统(成对的寡核苷酸引物和内部荧光TaqMan探针)，以及完成对研究标本的实际分析。核心设施的可用性完成了对常见生物系统(例如，OTC和MPS VII小鼠模型的基因和基因产品)的分析，消除了冗余，并提供了更高水平的质量保证。这项新的定量技术还为“安全分析”(如慢病毒载体RCR)提供了灵敏的方法，比现有的培养技术更快、更可靠、更便宜。该技术还提供了一种定量基因产物(RT-PCR)的方法，该方法不存在现有的检测方法，或者用于不产生翻译或转录最终产物的DNA序列(转座子载体可能需要)。

The purpose of the Real-Time Quantitative PCR Core is to assist investigators in evaluating gene transfer efficiency and transgenic expression in target ells for projects in the Gene Therapy for Metabolic Disorders program. As initially demonstrated by the current gene therapy clinical trial ("Lymphocyte gene therapy for Hunter syndrome), this technique fills a very significant scientific need to accurately quantitate the presence of a gene, and to measure its expression by reverse transcriptase-PCR (RT-PCR) with a high level of sensitivity and reproducibility. This core will assist investigators in designing assay systems (paired oligonucleotide primers and an internal fluorescent TaqMan probe), as well as accomplish the actual assays of research specimens. The availability of a core facility accomplishing assays for common biologic systems (e.g., gene and gene-products of OTC and MPS VII mouse models) eliminates redundancy and provides a greater level of quality assurance. The new, quantitative technology also provides sensitive methods for "safety assays" (e.g., for lentiviral vector RCR) that are much faster, reliable and inexpensive that existing culture techniques. The techniques also provides a method for quantitating gene products (RT-PCR) for which there are no existing assays, or for DNA sequences that do not yield a translational or transcriptional end product (as might be needed for transposon vectors).