CORE--MOLECULAR GENETICS CORE

分子遗传学核心旨在提供技术支持 给参与这个合作项目的各个实验室， 促进在以下方面开发的强大新工具的应用 第一个资金周期。总结现有的可用技术 对于弓形虫来说，现在可以表达重组分子 转化体，转化体 整合到基因组中，并以单拷贝或多拷贝的形式 转基因。稳定的集成可以通过随机方式进行 非同源重组、单位同源重组 (在转染区产生复制)或作为完美的 等位基因替换(或基因敲除)。这些方面中的许多方面可以 通过使用适当的矢量、可选择的标记和 转换条件。总而言之，现在可以应用 几乎所有的分子遗传工具都可以 弓形虫。 观察到的稳定转变的极高频率 (高达6%的可存活的转染速殖子)允许各种 重要的应用。基因克隆技术的可行性研究 互补作用可能有助于确定耐药基因座 作用机制未知的杀寄生剂(RoOS)。 插入突变技术已被用于标记腺苷 KEK、HXGPRT和UPRT基因座(Joiner、Ullman)，导致 成功克隆HXGPRT和UPRT cDNAs(AK应随后 不久)。这些技术还提供了替代方案 用于识别未知目标(RoOS)的互补性。基因 击倒提供了一种直接的手段来验证有希望的目标为 寄生虫生存所必需的(Joiner，Ullman)和等位基因 替换允许对分子模型进行关键评估 转基因寄生虫(乌尔曼)。 还将努力进一步发展 分子工具(更好、更安全的可选标记， 运行突变载体、表体文库和穿梭载体)和 试剂(包括不同阶段的特定克隆和文库 寄生虫菌株)。最后，因为这些技术带来了 前所未有的权力，调查弓形虫，与 NCDDG研究的有针对性的“国家合作”性质，我们 将继续协助技术转让和与 世界各地的其他组织，当这种合作可能 此外，改善治疗选择的最终目标是消除 或减轻艾滋病弓形虫病的负担。

The molecular genetics core is intended to provide technical support to the various laboratories involved in this cooperative project, facilitating application of the powerful new tools developed during the first funding cycle. To summarize existing technology available for Toxoplasma, it is now possible to express recombinant molecules both transiently and as stable transformants, as episomes or integrated into the genome, and as single copy or multicopy transgenes. Stable integration can arise either by random nonhomologous recombination, single site homologous recombination (producing a duplication at the transfected locus) or as a perfect allelic replacement (or gene knockout). Many of these aspects can be controlled by the use of appropriate vectors, selectable markers, and transformation conditions. In sum, it is now possible to apply virtually the entire spectrum of molecular genetic tools to Toxoplasma. The extraordinarily high frequency of stable transformation observed (up to 6% of viable transfected tachyzoites) permits a variety of important applications. The feasibility of gene cloning by complementation may allow identification of drug resistance loci for parasiticidal agents whose mechanism of action is unknown(Roos). Insertional mutagenesis has already been used to tag the adenosine kinase, HXGPRT, and UPRT loci (Joiner, Ullman), leading to the successful cloning of HXGPRT and UPRT cDNAs (AK should follow shortly). These techniques also provide an alternative to complementation for the identification of unknown targets (Roos). Gene knock-outs supply a direct means to validate promising targets as essential for parasite survival (Joiner, Ullman), and allelic replacement permits critical assessment of molecular models in transgenic parasites (Ullman). Efforts will also be directed toward the development of further molecular tools (better - and safer - selectable markers, 'hit-and- run' mutagenesis vectors, episomal libraries and shuttle vectors) and reagents (including stage-specific clones and libraries for different parasite strains). Finally, because these techniques bring unprecedented power to the investigation of T. gondii, in keeping with the targeted 'National Cooperative' nature of the NCDDG research, we will continue to assist in technology transfer and collaboration with other groups around the world, when such cooperation is likely to further the ultimate goal of improved treatment options to eliminate or alleviate the burden of AIDS toxoplasmosis.