PARENT SESSION
NUCLEAR TRANSFER, CLONING, AND REPRODUCTIVE TECHNOLOGIES
Harborside C
7:30 AM-10:00 AM

(348) EFFECT OF THE TIMING OF OOCYTE ACTIVATION ON RAT CLONING USING NEURAL STEM CELL NUCLEI.

Roh, Sangho^1,2, Du, Zhongtao^1,2, Fida, Shahnaz^1,2, Handford, Cheryl^1,2, Malakooti, Nakisa^1,2, Morrison, John^1,2, Trounson, Alan^1,2, ¹ Monash Institute of Reproduction and Development, Clayton, Australia² CopyRat Pty. Ltd., Clayton, Australia

ABSTRACT- To date, attempts to produce rats from nuclear transfer (NT) using fetal fibroblasts and cumulus cells have not yet been successful. We have examined methods for activation of rat oocytes, nuclear injection and activation of reconstructed eggs for the production of NT rat embryos from fetal neural stem cells. Neural stem cells were isolated from Day 14.5 fetuses in neurobasal media (Gibco) in the presence of bFGF (10 ng/ml) and EGF (10 ng/ml). The oocytes were recovered from 4-wk old Sprague Dawley rats treated with eCG and hCG injection in the HEPES-buffered medium supplemented with 300 IU/ml hyaluronidase and 1 g/ml nocodazole. The enucleation was carried out by slitting the zona pellucida in the region of the cytoplasmic bulge using a microneedle with subsequent squashing of the metaphase plate through the slit with the holding pipette. The enucleated eggs were then placed in rat embryo culture medium (mR1ECM) before nuclear injection. Nuclei were injected into enucleated oocytes using pipettes drawn to an approximate inner diameter of 5 m, which were back-loaded with mercury and coated with 5% polyvinylpyrrolidone (PVP) immediately before use. The pipette with isolated nucleus was put into zona pellucida through the slit and advanced between half and three-quarters of the way through the oocyte. Piezoelectric actuation was used to break the membrane and the nucleus was deposited. Reconstructed oocytes were then immediately exposed to activation medium of 10 mM SrCl₂ (with or without 5 g/ml cytochalasin B) for 4 h (Immediate activation methods; Group A: +cytochalasin B, and Group A′: -cytochalasin B), or cultured in mR1ECM for 2-3 h before activation treatment (Post-activation method; Group B: +cytochalasin B). Following this activation the oocytes were cultured in mR1ECM at 37°C in 5% CO₂ in air. Formation of pronucleus was checked the following morning and the development were recorded after 40 and 115 h of culture. In all experimental groups, 62.2-70.8% of reconstructed embryos showed clear structure of pronucleus following activation and 33.9-45.1% of the embryos developed over the 2-cell stage. These results show that immediate activation method can be used for rat nuclear transfer embryo production.

KEY WORDS: rat, nuclear transfer, neural stem cell, activation