Core--Real-time quantitative PCR facility

实时定量PCR核心的目的是帮助研究人员评估基因转移效率和靶细胞中的转基因表达，用于代谢紊乱基因治疗项目。正如目前的基因治疗临床试验（“Hunter综合征的淋巴细胞基因治疗”）最初所证明的，该技术满足了非常重要的科学需求，即准确定量基因的存在，并通过逆转录酶-PCR（RT-PCR）以高水平的灵敏度和再现性测量其表达。该核心将协助研究人员设计检测系统（配对寡核苷酸引物和内部荧光TaqMan探针），以及完成研究标本的实际检测。核心设施的可用性，完成对常见生物系统的测定（例如，OTC和MPS VII小鼠模型的基因和基因产物）消除了冗余，并提供了更高水平的质量保证。新的定量技术还提供了敏感的“安全测定”方法（例如，对于慢病毒载体RCR），其比现有培养技术快得多、可靠得多且便宜得多。该技术还提供了一种用于定量基因产物（RT-PCR）的方法，对于该方法，没有现有的测定法，或者对于不产生翻译或转录终产物（如转座子载体可能需要的）的DNA序列。

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).