Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.
The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently, we elucidated by using round spermatids that, after nuclear transfer, treatment of zygotes with trichostatin A (TSA), an inhibitor of histone deacetylase, can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami, N. Van Thuan, T. Hikichi, H. Ohta, S. Wakayama. E. Mizutani, T. Wakayama, Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids, Dev. Biol. (2005) in press]. Here, we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells, spleen cells, neural stem cells, and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further, we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus, our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques.
Kishigami
Satoshi
S
Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan. kishigami@cdb.riken.jp
Mizutani
Eiji
E
Ohta
Hiroshi
H
Hikichi
Takafusa
T
Thuan
Nguyen Van
NV
Wakayama
Sayaka
S
Bui
Hong-Thuy
HT
Wakayama
Teruhiko
T
D000818:Animals; D001755:Blastocyst / Q000187:drug effects*; D002467:Cell Nucleus / Q000187:drug effects; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005260:Female; D006877:Hydroxamic Acids / Q000008:administration & dosage*; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects*
D006877:Hydroxamic Acids; C012589:trichostatin A