Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.
The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.
Yang
Feikun
F
Department of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians University Munich, Hackerstrasse 27, 85764 Oberschleissheim, Germany.
Hao
Ru
R
Kessler
Barbara
B
Brem
Gottfried
G
Wolf
Eckhard
E
Zakhartchenko
Valeri
V
D000107:Acetylation; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D020148:Butyric Acid / Q000494:pharmacology; D002678:Chimera; D047108:Embryonic Development; D044127:Epigenesis, Genetic; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism; D008677:Metaphase; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D010312:Parthenogenesis; D011247:Pregnancy; D011256:Pregnancy Outcome; D011817:Rabbits
D056572:Histone Deacetylase Inhibitors; D006657:Histones; D020148:Butyric Acid