DNA hypomethylation of karyoplasts for bovine nuclear transplantation.
The objective of this research was to evaluate if DNA hypomethylation in cells used as karyoplasts would improve development of bovine nuclear transplantation (NT) embryos. DNA from serum-fed (SF), serum-starved (SS), and 1, or 5 microM 5-azacytidine (5-aza-CR) treated cells was digested with a methylation sensitive enzyme, and evaluated for DNA methylation. A significant reduction in DNA methylation was observed in cells cultured for 48 or 72 hr in SS medium as well as in cells cultured for 48 hr in the presence of 5 microM 5-aza-CR when compared to cells cultured in SF medium. All other comparisons contained no significant differences when compared to controls. When donor cells were cultured in 5-aza-CR, SF, or SS treatment media for 48 hr, no significant difference was observed (P = 0.06) in blastocyst development rates after NT. One embryo produced by donor cells treated with 5-aza-CR established a pregnancy. Four pregnancies resulted from embryos produced by SS donor cell NT and 3 resulted from embryos produced by SF donor cell NT. Supplementation of the donor cell culture medium with 5-aza-CR was not beneficial for increasing blastocyst rate or establishing pregnancy after NT.
Jones
K L
KL
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas, USA. kljones@siu.edu
Hill
J
J
Shin
T Y
TY
Lui
L
L
Westhusin
M
M
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects; D002478:Cells, Cultured; D019976:Cloning, Organism; D016895:Culture Media, Serum-Free; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D005314:Embryonic and Fetal Development; D005260:Female; D005347:Fibroblasts / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D011247:Pregnancy
D000964:Antimetabolites, Antineoplastic; D016895:Culture Media, Serum-Free; D004247:DNA; D001374:Azacitidine