Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo.
As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.
Nakamura
Kaori
K
Laboratory of Develomental and Reproductive Biotechnology, Department of Animal Resource Sciences, School of Veterinary Medicine and Life Sciences, Azabu University, Sagamihara, Japan.
Murata
Chisato
C
Ito
Masanori
M
Iwamori
Tokuko
T
Nishimura
Seiichiro
S
Hisamatsu
Shin
S
Sonoki
Shigenori
S
Nakayama
Aya
A
Suyama
Eigo
E
Kawasaki
Hiroaki
H
Taira
Kazunari
K
Nishino
Koichiro
K
Tachi
Chikashi
C
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D024282:Genes, sry; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects*; D012366:RNA, Transfer, Val; D012744:Sex Ratio
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012366:RNA, Transfer, Val; C427204:hammerhead ribozyme