Reproductive Technology

Cattle Cloned from Increasingly Differentiated Muscle Cells

Abstract
It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. In order to test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for one to six days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for one, four and five days were classified as myogenic precursors (MPC), myotubes (MT) and muscle-derived fibroblasts (MF) with a purity of 92%, 85% and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in i) cells selected for NT, ii) metaphase II oocytes, iii) NT couplets, iv) NT reconstructs, v) NT 2-cell embryos, and vi) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, while housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118/520 = 23% vs. 93/873 = 11% vs. 66/174 = 38% for MPC vs. MT vs. MF, respectively, P<0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (14/22 = 64% vs. 8/23 = 35% vs. 10/22 = 45% and 4/22 = 18% vs. 2/23 = 9% vs. 3/22 = 14%, for MPC vs. MT vs. MF, respectively).

Key words: Embryo • Assisted Reproductive Technology • Developmental biology • Early development