PARENT SESSION
REPRODUCTIVE TECHNOLOGIES

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(638) AGGREGATING NUCLEAR TRANSFER RECONSTRUCTS INCREASES INNER CELL MASS-TO-TOTAL CELL RATIO AND DOES NOT IMPROVE SOMATIC CATTLE CLONING EFFICIENCY.

Oback, Björn¹, Eichenlaub, Michael ¹, Laible, Götz¹, Tucker, Fleur ¹, Miller, Andria¹, Oliver, Jan¹, Troskie, Hilda¹, Forsyth, Jacqui¹, Berg, Martin¹, McMillan, Vicki¹, Wells, David¹, ¹ Reproductive Technologies, AgResearch Ltd., Hamilton, New Zealand

ABSTRACT- Inadequate epigenetic reprogramming of the somatic donor cell genome may be the major cause for low cloning efficiencies using nuclear transfer (NT). We aimed to improve cloning efficiency by aggregating bovine NT reconstructs. In mouse NT embryos, a similar epigenetic complementation approach resulted in normalized Oct4 gene expression and higher rates of in vivo development. Using our standard operating procedure for zona-free NT, we reconstructed cloned embryos from nine primary cell lines: 1) ear skin fibroblasts (passage 2-5) from six different adult bulls; 2) lung fibroblasts (passage 3-8) from a day 60 bovine female fetus (BFF) and 3) two lines of genetically modified BFF cells (passage 11-15) carrying copies of the human myelin basic protein. For NT, presumptive G0 cells were cultured in medium containing 0.5% FCS for 3-7 days. During activation and in vitro culture (IVC), reconstructed embryos were kept either singularly (single-IVC) or as aggregates of three (triple-IVC). In vitro fertilized (IVF) embryos cultured in single-and triple-IVC served as controls. After seven days, grade 1 and 2 blastocysts were differentially stained with propidium iodide and Hoechst 33342 following standard procedures. Blue and pink colours under epifluorescence were designated as inner cell mass (ICM) and trophectoderm (TE) cells, respectively, and counted. The total cell (TC) number was not different between IVF and NT embryos from triple- vs. single-IVC (243 and 207 vs. 113 and 116 cells, respectively). However, the ICM/TC ratio was increased in both IVF and NT embryos from triple- vs. single-IVC (0.56 and 0.56 vs. 0.34 and 0.44 with P<0.005, respectively). For single-IVC, the ratio were increased in NT vs. IVF embryos (0.78 and 0.44 vs. 0.51 and 0.34 with P<0.0001 and P<0.001, respectively), whereas embryos from triple-IVC had similar ratios. Across all nine cell lines, pregnancy establishment at D35, and development to term or weaning was not different between NT embryos from single- and triple-IVC (52/119 = 44% vs. 50/113 = 45%, 16/119 = 13% vs. 19/113 = 17% and 10/119 = 8% vs. 8/113 = 7%, respectively). Average birth weights of single- and triple-IVC cloned calves were very similar (43.1 vs. 43.9 kg, respectively). Survival beyond weaning was not different; so far all calves appear healthy and are now 10-18 months old.

KEY WORDS: early development, reproductive technology, nuclear transfer, pregnancy