CORE--MOLECULAR GENETICS CORE

分子遗传学核心旨在提供技术支持 参与这个合作项目的各个实验室， 促进应用在此期间开发的强大的新工具， 第一个融资周期。总结现有技术 对于弓形虫，现在可以表达重组分子， 瞬时和作为稳定转化体，作为附加体或 整合到基因组中，并作为单拷贝或多拷贝 转基因稳定的整合可以通过随机的 非同源重组，单位点同源重组 （在转染的基因座处产生重复）或作为完美的 等位基因替换（或基因敲除）。其中许多方面可以 通过使用适当的载体、选择性标记和 转化条件总之，现在可以应用 几乎所有的分子遗传学工具 弓形虫 观察到的异常高的稳定转化频率 (up至6%的活的转染速殖子）允许多种 重要应用。基因克隆的可行性 互补可允许鉴定用于治疗癌症的药物抗性基因座。 作用机制未知的杀寄生虫剂（鲁什）。 插入突变已经用于标记腺苷 激酶、HXGPRT和UPRT基因座（Joiner，Ullman），导致 成功克隆HXGPRT和UPRT cDNA（AK应遵循 不久）。这些技术还提供了一种替代方案， 用于鉴定未知靶标的互补（鲁什）。基因 敲除提供了一种直接的手段来验证有前途的目标， 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（Ullman）。 还将努力进一步发展 分子工具（更好和更安全的选择标记，“命中和” 运行诱变载体、附加型文库和穿梭载体）和 试剂（包括不同阶段特异性克隆和文库） 寄生虫株）。最后，因为这些技术带来了 对T.弓形虫，与 鉴于NCDDG研究具有针对性的“国家合作”性质，我们 将继续协助技术转让和合作， 世界各地的其他团体，当这种合作可能会 进一步实现改善治疗方案的最终目标，以消除 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.