PROTEIN PAINTING OF PREIMPLANTATION EMBRYOS

植入前胚胎的蛋白质涂色

• 批准号: 6638023
• 负责人: CAROL M WARNER
• 金额: $ 24.96万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638023
• 关键词: T lymphocyte; affinity chromatography; animal birth weight; biotechnology; cell membrane; crosslink; detergents; developmental genetics; embryo /fetus; embryo implantation; embryogenesis; gene expression; gestational age; glycosylphosphatidylinositols; laboratory mouse; monoclonal antibody; protein engineering; protein purification; protein structure function

DESCRIPTION: (Scanned from the applicant's abstract) The rate of growth of preimplantation embryos is regulated by both environmental and genetic factors and can have a profound effect on the later health of the offspring from birth into adulthood. Using the mouse as a model system, one gene that influences growth rate of preimplantation embryos and pregnancy outcome has been identified, the Ped gene. The product of the Ped gene is Qa-2 protein. This protein is attached to the plasma membrane of embryonic cells by a glycosylphosphatidylinositol (GPI) linkage. Embryos with a fast embryonic growth rate (Pedfast) express GPI-linked Qa-2 protein and have a greater chance of survival to term, as well as higher birth and weaning weights compared to embryos with the absence of GPI-linked Qa-2 protein (Ped slow). Proteins with a GPI linkage can spontaneously incorporate into the plasma membrane of somatic cells (e.g., T cells) in a procedure called "protein painting." In this proposal GPI-linked Qa-2 protein will be used to evaluate whether protein painting can be utilized to add proteins to the cell surface of preimplantation embryos. First, GPI-linked Qa-2 protein will be purified by detergent solubilization followed by affinity chromatography with anti-Qa-2 monoclonal antibody. Next, the purified GPI-linked Qa-2 protein will be used to optimize conditions for painting of preimplantation embryos. Then, the function of the painted Qa-2 protein during the preimplantation period will be assessed by measuring rate of growth and response to crosslinking with anti-Qa-2 antibody. Finally, the effect of the modification of the embryos during the preimplantation period on pregnancy outcome will be determined by evaluating survival to term, birth weight, and weaning weight. Protein painting is a potentially powerful technology because cell surface protein expression can be manipulated without gene transfer. Moreover, any protein could be used for protein painting by splicing the signal to add a GPI anchor to the gene encoding the protein of interest. Protein painting may ultimately be applied to create better conditions for modulating preimplantation embryonic growth or death, leading to enhanced pregnancy outcome or a new contraceptive technology.

描述：（从申请人的摘要扫描）的增长率 植入前胚胎受环境和遗传因素的调控 并对后代出生后的健康产生深远的影响 长大成人使用小鼠作为模型系统， 着床前胚胎的生长速度和妊娠结局 发现了Ped基因。Ped基因的产物是Qa-2蛋白。这 蛋白质通过一种 糖基磷脂酰肌醇（GPI）键。快速胚胎的胚胎 生长速率（Pedfast）表达GPI连接的Qa-2蛋白， 的存活率，以及更高的出生和断奶体重相比， 不存在GPI连接的Qa-2蛋白的胚胎（Ped slow）。蛋白与 GPI连接可以自发地整合到体细胞的质膜上， 细胞（例如，T细胞）在一个称为“蛋白质绘画”的过程中。“在这个 建议GPI连接的Qa-2蛋白将用于评估蛋白质是否 可以利用涂抹将蛋白质添加到植入前的细胞表面， 胚胎首先，将GPI连接的Qa-2蛋白通过洗涤剂纯化， 溶解后用抗Qa-2单克隆抗体亲和层析 抗体的接下来，纯化的GPI连接的Qa-2蛋白将用于优化 植入前胚胎的着色条件。然后， 将通过以下方法评估植入前期涂敷的Qa-2蛋白 测量生长速率和对抗Qa-2抗体交联的响应。 最后，对胚胎发育过程中的修饰作用进行了研究。 将通过评估植入前阶段对妊娠结局的影响来确定 足月存活率、出生体重和断奶体重。蛋白质绘画是一种 潜在的强大技术，因为细胞表面蛋白表达可以 不需要基因转移此外，任何蛋白质都可以用于 通过拼接信号将GPI锚加到基因上， 编码感兴趣的蛋白。蛋白质绘画最终可能被应用于 为调节植入前胚胎生长创造更好的条件， 死亡，从而提高怀孕结果或新的避孕技术。