Gene expression and in vitro development of inter-species nuclear transfer embryos.
This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.
Arat
Sezen
S
Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602, USA.
Rzucidlo
S Jacek
SJ
Stice
Steven L
SL
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000235:genetics; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000254:growth & development; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D014158:Transcription, Genetic / Q000235:genetics
D012333:RNA, Messenger