Rocky Mountain Neurological Disorders Core

C2核心B;基因靶向/病毒载体核心 C2.1基本原理。小鼠的主动遗传操作和神经组织中蛋白质表达的病毒靶向已经导致神经系统研究的革命。尽管对涉及小鼠遗传操作的实验的解释必须小心进行，但在修饰单个基因后在体内监测CNS变化的能力使该技术成为现代研究的不可或缺的工具。 神经生物学基因改变的小鼠的有用性将随着基因靶向小鼠的使用增加而进一步增长，所述基因靶向小鼠表达缺乏特定蛋白质-蛋白质相互作用的蛋白质、条件性敲除、BAC转基因和表达外部触发的神经元开关的动物。使用病毒载体来急剧改变蛋白质表达是一种与遗传操作互补的强大技术。 UCD AMC的NINDS研究人员正在使用各种基因改变的小鼠和病毒载体来研究神经生物学中的基本问题。虽然我们可以使用Charles C Gates再生医学和干细胞生物学项目的转基因设施来生产基因靶向小鼠，但进入点是完成的转基因载体或转染的ES细胞（参见附录，Peter博士的信 Koch）。此外，如果核心B不存在，则必须由个体研究者通过PCR和/或Southern印迹法进行ES克隆筛选-所有劳动密集型程序。对于转基因小鼠，对于注射的每个构建体获得几个品系。然后需要对这些细胞系进行初步测试，以确定它们是否整合了外源DNA。随后，分析适当的组织特异性转基因表达。 必要的，以及生殖系传播的分析。这些ES和转基因研究都需要分子生物学和小鼠饲养方面的技能，而这些技能不是大多数研究人员的专业领域。 同样，病毒载体的构建和生产也需要特定的专业知识，这在大多数实验室中并不容易单独建立。核心B为RMNDC用户提供这些专门服务。

C2 Core B; Gene-Targeting/Viral Vector Core C2.1 Rationale. Active genetic manipulation of mice and viral targeting of protein expression in neural tissue have resulted in a revolution in the study of the nervous system. Although interpretation of experiments involving genetic manipulation of mice must be performed with care, the ability to monitor changes in the CNS in vivo upon modification of a single gene makes this technique an indispensable tool for modern inquiry in neurobiology. The usefulness of genetically altered mice will grow even further with increased use of genetargeted mice expressing proteins deficient for specific protein-protein interactions, conditional knockouts, BAC-transgenics and animals expressing externally-triggered neuronal switches. Use of viral vectors to acutely alter protein expression is a powerful technique complementary to genetic manipulation. The group of NINDS researchers at the UCD AMC is using a variety of genetically altered mice and viral vectors to study fundamental questions in neurobiology. Although we have access to a the transgenic facility of the Charles C Gates Regenerative Medicine and Stem Cell Biology Program to produce gene targeted mice, the point of entry is a finished transgenic vector or transfected ES cells (see Appendix, letter from Dr. Peter Koch). Moreover, if Core B did not exist ES clone screening by PCR and/or Southern blotting - all labor intensive procedures - would have to be performed by the individual investigator. For transgenic mice, several lines are obtained for each construct injected. These lines then need to be tested initially to see if they have integrated the exogenous DNA. Subsequently, analysis of appropriate tissue-specific transgene expression is necessary, as well as the analysis of germ-line transmission. These ES and transgenic studies all require skills in molecular biology and mouse husbandry that are not the areas of expertise of the majority of investigators. Likewise, construction and production of viral vectors also requires specific expertise that is not easily established in most laboratories individually. Core B provides these specialized services to RMNDC users.