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.

[1]  D. Lilley,et al.  Structure, folding and mechanisms of ribozymes. , 2005, Current opinion in structural biology.

[2]  K. Taira,et al.  Gene discovery by ribozyme and siRNA libraries , 2005, Nature Reviews Molecular Cell Biology.

[3]  Hiroaki Kawasaki,et al.  Transcriptional gene silencing by short interfering RNAs. , 2005, Current opinion in molecular therapeutics.

[4]  J. Uney,et al.  RNA interference and the use of small interfering RNA to study gene function in mammalian systems. , 2004, Journal of molecular endocrinology.

[5]  Hiroaki Kawasaki,et al.  World of small RNAs: from ribozymes to siRNA and miRNA. , 2004, Differentiation; research in biological diversity.

[6]  V. Harley,et al.  Turning on the male – SRY, SOX9 and sex determination in mammals , 2003, Cytogenetic and Genome Research.

[7]  G. Krupp,et al.  Ribozyme Biochemistry and Biotechnology , 2000 .

[8]  J. Doudna,et al.  Ribozyme structures and mechanisms. , 2000, Annual review of biochemistry.

[9]  Phillip D. Zamore,et al.  RNA Interference , 2000, Science.

[10]  C. Hunter,et al.  Gene silencing: Shrinking the black box of RNAi , 2000, Current Biology.

[11]  C. Shelburne,et al.  Inhibition of kit expression in P815 mouse mastocytoma cells by a hammerhead ribozyme. , 1999, Clinical immunology.

[12]  S. Asano,et al.  Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III , 1999, Journal of Virology.

[13]  D. Stinchcomb,et al.  Anti-gene therapy: the use of ribozymes to inhibit gene function. , 1996, Trends in genetics : TIG.

[14]  Satoshi Tanaka,et al.  Effects of Transfected Human and Mouse SRY/Sry Genes upon the Expression of Hypothetical SRY-Cascade Genes in Cultured Mouse Sertoli Cell Line, TM-4 , 1996 .

[15]  M. Komatsu,et al.  Specific regulation of gene expression by antisense nucleic acids: a summary of methodologies and associated problems. , 1996, Artificial organs.

[16]  Satoshi Tanaka,et al.  Quantitative Analysis of the Expression of Whey Acidic Protein (WAP) mRNA in the Prolactin-Responsive Mouse Mammary Gland Epithelial Cell Line, HC11 , 1995 .

[17]  K. Miyagawa,et al.  Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing , 1995, Cell.

[18]  K. Taira,et al.  Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. , 1995, Biochemistry.

[19]  P. Goodfellow,et al.  Inverted repeat structure of the Sry locus in mice. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[21]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[22]  G. Schaffner,et al.  Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro , 1989, Molecular and cellular biology.

[23]  G. Hannon.  RNA interference : RNA , 2002 .

[24]  S. Crooke,et al.  Progress in antisense technology: the end of the beginning. , 2000, Methods in enzymology.

[25]  B. Capel.  Sex in the 90s: SRY and the switch to the male pathway. , 1998, Annual review of physiology.

[26]  P. Koopman,et al.  The molecular biology of SRY and its role in sex determination in mammals. , 1995, Reproduction, fertility, and development.

[27]  P N Goodfellow,et al.  SRY and sex determination in mammals. , 1993, Annual review of genetics.