Core B: FSH Glycoform Production, Purification, and Analysis

项目概要/摘要： 核心B - FSH糖型的生产、纯化和分析 核心B提供关键和必要的试剂、分析和专业知识，以支持所有项目的实施 他们的具体目标。具体而言，核心B将继续生产重组hFSH糖型和突变hFSH 糖型已经建立了从葡萄糖中产生毫克量的糖型变体的方法。 用人FSH a和B亚基基因、CGA和CGA转染培养的GH 3细胞的条件培养基， FSHB或突变FSHB基因。核心B将继续开发转染系统，以改善 突变hFSH糖型生产，为所有四个项目提供激素。这些hFSH变体模拟已知的 重组低糖基化hFSH糖型对卵巢细胞的作用。核心B将继续 与项目4（Bousfield）合作，产生和评估特异性识别完整hFSH的抗体， hFSH a亚基以及单个糖型B亚基，用于糖型分离和检测。核心B 将为项目2（戴维斯）、项目3（乔纳斯）和项目4（鲍斯菲尔德）提供各种电池，以评估 糖型受体结合，FSH受体寡聚化，FSH受体介导的信号转导和 糖型影响细胞分化。待提供的细胞包括KGN细胞，一种人颗粒细胞系 表达人FSH受体的CHO细胞;和原代猪FSH受体。 天然表达FSH受体的颗粒细胞来完成正在进行的实验。GH 3和HEK 293 用hFSH和突变基因转染的细胞对于重组hFSH糖型的产生是关键的。 核心B将为项目提供分析支持，包括：FSH RIA和ELISA，以监测hFSH水平 在FSH糖型产生期间和各种生物体液（包括血清和尿液）中;小鼠FSH ELISA 对于项目1（Kumar）; FSH和LH受体结合;孕酮ELISA监测hFSH变体对 靶细胞功能; hFSH变体抗原ELISA用于单克隆抗体筛选。核心B将获得和 利用猪卵丘卵母细胞复合体（pCOCs）开展研究，以评估 COC扩增和基因表达后的hFSH糖型变体。

Project Summary/Abstract: Core B – FSH Glycoform Production, Purification and Analysis Core B provides critical and requisite reagents, analyses, and expertise to support all of the Projects in fulfilling their Specific Aims. Specifically, Core B will continue to produce recombinant hFSH glycoforms and mutant hFSH glycoforms. Procedures have been established to produce milligram quantities of glycoform variants from the conditioned medium of cultured GH3 cells transfected with the human FSH a and b subunit genes, CGA and FSHB, respectively, or mutant FSHB genes. Core B will continue to develop transfection systems to improve mutant hFSH glycoform production to supply hormone to all four Projects. These hFSH variants mimic known recombinant hypoglycosylated hFSH glycoforms in terms of their actions upon ovarian cells. Core B will continue to work with Project 4(Bousfield), to generate and assess antibodies that recognize specifically intact hFSH, hFSH a subunit, as well as individual glycoform b subunits, for use in glycoform isolation and detection. Core B will provide a variety of cells for use in Project 2(Davis), Project 3(Jonas), and Project 4(Bousfield) to assess glycoform receptor binding, FSH receptor oligomerization, FSH receptor mediated signal transduction and glycoform impact upon cell differentiation. Cells to be furnished include KGN cells, a human granulosa cell line that responds to hFSH stimulation; CHO cells that express the human FSH receptor; and primary porcine granulosa cells which naturally express the FSH receptor to complete on-going experiments. GH3 and HEK293 cells transfected with hFSH and mutated genes are critical for the production of recombinant hFSH glycoforms. Core B will provide analysis support for the Projects including: FSH RIA and ELISA to monitor hFSH levels during FSH glycoform production and in various biological fluids including serum and urine; mouse FSH ELISA for Project 1(Kumar); FSH and LH receptor binding; progesterone ELISA to monitor hFSH variant impact upon target cell function; and hFSH variant antigen ELISA for monoclonal antibody screening. Core B will obtain and develop studies utilizing porcine cumulus oocyte complexes (pCOCs) to assess potential differential activities of hFSH glycoform variants upon COC expansion and gene expression.