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. ? ? ?
| Yurttas, Piraye; Vitale, Alejandra M; Fitzhenry, Robert J et al. (2008) Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo. Development 135:2627-36 |
