TARGETING SERUM BINDING PROTEINS IN DEVELOPMENT

这项申请的长期目标是彻底和深入 分子生物学的培训，将准备格伦格哈德博士， 未来独立的基础研究衰老的分子机制。 第一阶段包括通过研究生课程、研讨会、 演示和教学，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 推进和补充研究培训。 教学计划是 在南希·库克博士的实验室里， 和斯蒂芬·利哈勃，医学和人类遗传学系。 的 具体的研究项目将集中在破坏维生素D 结合蛋白（DBP/GC球蛋白）基因， 胚胎干细胞（ES细胞）中的同源重组。 DBP是最古老的 多基因家族成员，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缺失， 广泛的筛查表明DBP功能对正常的 发展 第一阶段研究的具体目标包括克隆和 表征小鼠DBP基因的一部分，构建一个 替换靶向载体，以及DBP-/DBP+ ES细胞的产生。 将在平行研究中靶向AFP基因。为了做到这一点，博士。 Gerhard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， 通过插入E.大肠杆菌新霉素 将抗性基因插入克隆的小鼠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。 ES细胞将电穿孔， 该构建体用G418和更昔洛韦双重选择。 双重药物 抗性菌落，反映了neo基因的整合和 选择胸苷激酶基因，并通过PCR进行分析， 成功靶向同源重组的Southern印迹 DBP-/DBP+ ES细胞。 在第二阶段研究计划中， 将细胞显微注射到C57 B1/6 J胚泡中并重新植入 移植到假孕的受体体内以产生嵌合体。 雄性嵌合体 然后与C57 B1/6 J雌性回交以评估生殖系 传输 回交的生殖系后代将是专性的 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-小鼠将包括评价维生素D代谢、肌动蛋白 结合研究和嗜中性粒细胞趋化性测定。 定点 通过转基因技术诱变DBP基因和/或进一步 还计划进行基因靶向。

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.