MITOCHONDRIA IN AGING

这一应用程序的长期目标是彻底和深入的 分子生物学方面的培训将为格伦·格哈德博士 未来对衰老分子机制的独立基础研究。 第一阶段包括通过研究生课程、研讨会、 讲授和教学，重点是基因表达和 发展。一个咨询委员会将负责监督格哈德博士的 推进和补充科研培训。这个说教项目是 围绕南希·库克博士实验室的一个研究项目而建 和斯蒂芬·利巴伯，医学和人类遗传学系。这个 具体的研究项目将集中在维生素D的破坏上 结合蛋白(DBP/GC球蛋白)基因打靶 胚胎干细胞中的同源重组。DBP是最古老的 多基因家族的成员，包括白蛋白(ALB)和 甲胎蛋白(AFP)，其多种功能包括转运 维生素D甾醇，肌动蛋白单体的高亲和力结合，以及在 中性粒细胞趋化性。未检测到DBP删除，尽管 广泛的筛查表明DBP功能对正常至关重要 发展。第一阶段研究的具体目标包括克隆和 鉴定小鼠DBP基因的一部分，构建一个 置换靶向载体，获得DBP-/DBP+ES细胞。 AFP基因将成为平行研究的目标。为了做到这一点，Dr。 格哈德将首先克隆并鉴定小鼠DBP基因的一部分 包含外显子2和3。他将构建一个靶向载体，用于 大肠杆菌新霉素插入的阳性-阴性药物选择 将抗性基因插入到克隆的小鼠DBP基因组片段的外显子中。 然后将单纯疱疹病毒胸苷激酶基因连接到 基因组片段的一端。胚胎干细胞将被电穿孔 此构建物与G418和更昔洛韦双选。双重药物 抗性克隆，反映了neo基因的整合和 胸苷激酶基因，将选择和分析，通过聚合酶链式反应和 成功靶向同源重组的Southern blotting DBP-/DBP+ES细胞。在第二阶段研究计划期间，有针对性的ES 将细胞显微注射到C57B1/6J囊胚中并重新植入 变成假孕受体以产生嵌合体。雄性嵌合体将 然后与C57B1/6J雌性回交以评估种系 变速箱。回交的种系后代将是专性的 DBP-/DBP+小鼠。将这些小鼠杂交将产生DBP-/DBP- 纯合子。根据表型，广泛的功能分析 DBP-/DBP-小鼠将包括对维生素D代谢、肌动蛋白的评估 结合研究和中性粒细胞趋化试验。站点定向 通过转基因技术和/或进一步诱变DBP基因 基因打靶也在计划中。

The long-term objective of this application is thorough and intensive training in molecular biology that will prepare Dr. Glenn Gerhard for future independent basic research into molecular mechanisms of aging. Phase I includes academic training through graduate courses, seminars, presentation, and teaching, with emphasis on gene expression and development. An advisory committee will serve to monitor Dr. Gerhard's progress and supplement the research training. The didactic program is built around a research project in the laboratories of Drs. Nancy Cooke and Stephen Liebhaber, Departments of Medicine and Human Genetics. The specific research project will focus on disruption of the vitamin D binding protein (DBP/GC globulin) gene by gene targeting through homologous recombination in embryonic stem (ES) cells. DBP is the oldest member of a multigene family which includes albumin (ALB) and alpha-fetoprotein (AFP), whose diverse functions include transport of vitamin D sterols, high affinity binding of actin monomers, and a role in neutrophil chemotaxis. A deletion of DBP has not been detected despite extensive screening suggesting DBP functions are critical to normal development. The specific aims for Phase I research include cloning and characterizing a portion of the mouse DBP gene, construction of a replacement targeting vector, and the generation of DBP-/DBP+ ES cells. The AFP gene will be targeted in parallel studies. To do this, Dr. Gerhard will first clone and characterize a portion of the mouse DBP gene containing exons 2 and 3. He will construct a targeting vector for use in positive-negative drug selection by insertion of the E. coli neomycin resistance gene into an exon of the cloned mouse DBP genomic fragment. The Herpes Simplex Virus thymidine kinase gene will then be ligated to one end of the genomic fragment. ES cells will be electroporated with this construct and doubly selected with G418 and gancyclovir. Dual drug resistant colonies, reflecting integration of the neo gene and loss of the thymidine kinase gene, will be selected and analyzed by PCR and Southern blotting for successfully targeted homologous recombinant DBP-/DBP+ ES cells. During the phase II research plan, the targeted ES cells will be microinjected into C57B1/6J blastocysts and reimplanted into pseudopregnant recipients to generate chimeras. Male chimeras will then be back-crossed to C57B1/6J females to assess germ-line transmission. Germ-line progeny of the back-crosses will be obligate DBP-/DBP+ mice. Intercrossing these mice will produce DBP-/DBP- homozygotes. Depending upon phenotype, extensive functional analysis of DBP-/DBP- mice will include evaluation of vitamin D metabolism, actin binding studies, and neutrophil chemotaxis assays. Site-directed mutagenesis of the DBP gene through transgenic techniques and/or further gene targeting is also planned.