[6] Hybrid arrest screening in oocytes

Publisher Summary This chapter describes the method of hybrid arrest screening in the oocyte, which is used in cloning of the gonadotropin-releasing hormone (GnRH) receptor. There are two cloning strategies in which the oocyte bioassay has been used, (i) expression cloning and (ii) cloning by hybrid arrest screening. For expression cloning, mRNAs are transcribed in vitro from a cyclic DNA (cDNA) library containing the receptor of interest. A receptor cDNA clone is identified by its expression in the oocyte following injection of the transcribed mRNA. The original pool of cDNA clones is purified by stepwise fractionations of the response-evoking cDNA mixture until a single clone is obtained. To achieve proper expression of a protein in the oocyte, injection of the entire coding sequence of the mRNA is required. In cases where multiple subunits are required for activity, such as in the case of the nicotinic Ach receptor, mRNA for all subunits must be present for the expression of a functional receptor, but this may not always be feasible from a given cDNA library. The second cloning strategy in which the oocyte bioassay is used is the technique of hybrid arrest screening.