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

(343) BEHAVIOR OF MOUSE NUCLEI TRANSFERRED INTO ENUCLEATED MII BOVINE OOCYTES.

Arat, Sezen^1,2, Rzucidlo, S Jacek³, Stice, Steven^1,3, ¹ Department of Animal and Dairy Science, Athens, GA² Research Institute for Genetic Engineering and Biotechnology, Kocaeli, TR³ Prolinia Inc, Athens, GA

ABSTRACT- This study examined the chromatin morphology and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into bovine ooplasm. Bovine oocytes, isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FCS), sodium pyruvate, penicillin/streptomycin, rIGF-1, bFSH, and bLH. Bovine embryonic fibroblasts (BEF) were obtained from 60-day-old fetus, and MEFs were derived from 14-day-old fetus. Prior to nuclear transfer (NT), all somatic cells were cultured in media with 0.5% serum for 3 days. After NT and fusion, NT units were activated using a combination of calcium ionophore, cytochalasin B and cycloheximide and cultured for 7 days in BARC media. At 6 h after activation, 70% and 20% of NT units reconstructed with MEF (MEF-NT) had one and two swelled pronuclei, respectively. After 72 h of culture, 62.5% of MEF-NTs was arrested at 8-cell stage, 31.2% reached the 2- to 4- cell stage, and only 6.2% had more than 8 blastomeres. No development to blastocyst was observed in MEF-NTs, whereas 20% of NTs reconstructed with BEF (BEF-NT) reached the blastocyst stage. In addition, in 20% of 8-cell MEF-NTs nuclei were not present in all blastomeres. The expression of selected genes (Hsp 70.1, Oct4, embryonic (e)AP, eIF, Hprt, -actin) which are normally expressed in 8-cell stage mouse embryos was examined in 8-cell MEF-NTs, 8-cell mouse embryo, enucleated bovine oocytes and MEFs using RT-PCR. The mRNA for Hsp 70.1 and eIF genes, normally expressed after embryonic genome activation, was detected in MEF-NTs and MEF, but not in normal mouse embryos. Actin mRNA was detected in MEF-NT, normal mouse embryos and MEF, whereas Hprt mRNA was found in normal mouse embryo and MEF but not in MEF-NTs. Expression of Oct4 and eAP, which normally are expressed in undifferentiated cells, was detected only in normal mouse embryos. Mouse nuclei transferred into bovine ooplasm can form pronucleus-like structures and initiate the development. Although expression of some genes indicating minor zygotic activation could be detected, the lack of some stage specific genes like Oct4 and eAP may be an evidence of incomplete reprogramming of MEF nucleus in bovine oocyte and a reason for the arrest of NT embryo development.

KEY WORDS: nuclear transfer, gene expression, interspecies