CONTRACEPTIVE POTENTIAL OF OOCTYE-RESTRICTED cPLA2g

描述（由申请人提供）：对编码~67 kDA（pi 5.7）蛋白的新cDNA的序列分析发现，我们命名为卵磷脂酶A2 g（ePLA 2g）的该基因与人胞质磷脂酶A2 g具有~56%的同一性。北方印迹分析发现ePLA 2 g的表达仅限于卵母细胞，因此可能使这种母体蛋白成为理想的避孕靶点。我们制备了高度特异性的ePLA 2g抗血清，并进行了间接免疫荧光分析，以研究ePLA 2g的亚细胞定位。有趣的是，我们发现，聚集的ePLA 2g动态搬迁从卵母细胞皮层的核膜在germinal囊泡破裂，从而表明这种假定的蛋限制性磷脂酶A2 g在膜重塑的可能作用。本研究的主要目的是利用在卵母细胞中表达ePLA 2g短发夹RNA（shRNA）的转基因小鼠模型研究ePLA 2g在卵母细胞成熟和受精过程中的功能。该项目的第一部分将由印度新德里国家免疫学研究所的Anil Suri博士执行。Suri博士在该项目中的作用将是首先产生含有功能性ePLA 2g shRNA序列的慢病毒载体。然后，他将在体外验证ePLA 2g/pLL3.7载体的功能。该项目的下一部分将涉及用表达ePLA 2g shRNA的慢病毒颗粒感染受精卵，以产生具有功能沉默的ePLA 2g基因的转基因小鼠。然后将研究来自这些ePLA 2g敲低动物的卵母细胞的表型，以确定该基因是否是卵母细胞成熟和/或受精所需的。这项工作将在纽约市康奈尔医学院遗传医学系Scott Coonrod博士的实验室进行。正式证明ePLA 2g是卵母细胞成熟和/或受精所必需的，使用shRNAi [短发夹RNA干扰]反向遗传方法将可能使该分子成为理想的避孕靶点。

DESCRIPTION (provided by applicant): Sequence analysis of a novel cDNA encoding a ~67 kDA (pi 5.7) protein found that this gene, which we named egg phospholipase A2g (ePLA2g), is ~56% identical to human cytosolic phospholipase A2g. Northern blot analysis finds that ePLA2g expression is restricted to the oocyte, thus potentially making this maternal protein an ideal contraceptive target. We generated highly specific ePLA2g antisera and performed indirect immunofluorescence analysis to investigate ePLA2g's subcellular localization. Interestingly, we found that aggregates of ePLA2g dynamically relocate from the oocyte cortex to the nuclear envelope during germinal vesicle breakdown, thus suggesting a possible role for this putative egg-restricted phospholipase A2g in membrane remodeling. The main goal of this research project is to investigate the function of ePLA2g during oocyte maturation and fertilization using a transgenic mouse model in which ePLA2g short hairpin RNAs (shRNA) are expressed in the oocyte. The first portion of this project will be performed by Dr. Anil Suri at the National Institute of Immunology in New Dehli, India. Dr. Suri's role in the project will be to first generate a lentiviral vector containing a functional ePLA2g shRNA sequence. He will then validate the functionality of the ePLA2g/pLL3.7 vector in vitro. The next portion of the project will involve infecting zygotes with lentiviral particles expressing the ePLA2g shRNAs in order to generate transgenic mice possessing a functionally silenced ePLA2g gene. The phenotype of oocytes from these ePLA2g knockdown animals will then be studied to establish if the gene is required for oocyte maturation and/or fertilization. This work will be performed in Dr. Scott Coonrod's laboratory in the Department of Genetic Medicine at Cornell Medical College in New York City. A formal demonstration that ePLA2g is required for oocyte maturation and/or fertilization using the shRNAi [short hairpin RNA interference] reverse genetic approach will likely make this molecule an ideal contraceptive target.