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BOR - Papers in Press, published online ahead of print May 23, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.058164
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BIOLOGY OF REPRODUCTION 77, 395–406 (2007)
DOI: 10.1095/biolreprod.106.058164
© 2007 by the Society for the Study of Reproduction, Inc.

Cattle Cloned from Increasingly Differentiated Muscle Cells1

A.L Green , D.N Wells , and B Oback 2

Ruakura Research Centre, Reproductive Technologies, AgResearch Ltd., Hamilton, New Zealand

ABSTRACT

It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1–6 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 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 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 (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).

assisted reproductive technology, developmental biology, early development, embryo

FOOTNOTES

1Supported by the New Zealand Foundation for Research, Science, and Technology and AgResearch.

Correspondence: 2FAX: 64 7 838 5536; e-mail: bjorn.oback{at}agresearch.co.nz






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Copyright © 2007 by the Society for the Study of Reproduction.