PROTEIN PAINTING OF PREIMPLANTATION EMBRYOS

描述：(摘自申请者的摘要)增长速度 植入前胚胎受环境和遗传因素的调节。 会对后代出生后的健康产生深远的影响 进入成年期。使用老鼠作为模型系统，一个基因影响 植入前胚胎的生长速度和妊娠结局 经鉴定，是Ped基因。Ped基因的产物是Qa-2蛋白。这 蛋白质通过一种蛋白质附着在胚胎细胞的质膜上 糖基磷脂酰肌醇(GPI)连接。具有快速胚胎的胚胎 Growth Rate(Pedfast)表达与GPI相关的Qa-2蛋白，有更大的机会 足月存活率，以及更高的出生和断奶体重 缺乏GPI连接的Qa-2蛋白的胚胎(Ped Slow)。蛋白质中含有一个 GPI连锁可以自发地结合到细胞膜上 细胞(例如，T细胞)在一种称为“蛋白质涂抹”的过程中。在这 建议使用GPI连接的Qa-2蛋白来评估蛋白质 涂抹可以用来将蛋白质添加到植入前的细胞表面 胚胎。首先，GPI连接的Qa-2蛋白将用洗涤剂提纯 增溶后的抗Qa-2单抗亲和层析 抗体。接下来，纯化的GPI连接的Qa-2蛋白将用于优化 植入前胚胎涂抹的条件。然后， 植入前期绘制的Qa-2蛋白将通过以下方式进行评估 用抗Qa-2抗体测定细胞的生长速度和对交联物的反应。 最后，在胚胎发育过程中对胚胎进行改造的效果 植入前时间对妊娠结局的影响将通过评估 存活至足月、出生体重和断奶体重。蛋白质绘画是一种 潜在的强大技术，因为细胞表面蛋白的表达可以 在没有基因转移的情况下被操纵。此外，任何蛋白质都可以用来 通过拼接信号将GPI锚点添加到基因中来绘制蛋白质 编码感兴趣的蛋白质。蛋白质涂装最终可能被应用于 为调控着床前胚胎生长创造更好的条件 死亡，导致改善妊娠结局或新的避孕技术。

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.