RESUMEN
Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.
Asunto(s)
Marcación de Gen , ARN Catalítico , Ribonucleasa P , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Humanos , Marcación de Gen/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ingeniería Genética/métodos , Citomegalovirus/genéticaRESUMEN
Infection by varicella-zoster virus (VZV) can be prevented by using live attenuated vaccines. VZV vaccine strains are known to evolve rapidly in vivo, however, their genetic and biological effects are not known. In this study, the plaque-purified vaccine strain Suduvax (PPS) was used to understand the genetic changes that occur during the process of propagation in in vitro cell culture. Full genome sequences of three different passages (p4, p30, and p60) of PPS were determined and compared for genetic changes. Mutations were found at 59 positions. The number of genetically polymorphic sites (GPS) and the average of minor allele frequency (MAF) at GPSs were not significantly altered after passaging in cell culture up to p60. The number of variant nucleotide positions (VNPs), wherein GPS was found in at least one passage of PPS, was 149. Overall, MAF changed by less than 5% at 52 VNPs, increased by more than 5% at 42 VNPs, and decreased by more than 5% at 55 VNPs in p60, compared with that seen in p4. More complicated patterns of changes in MAF were observed when genetic polymorphism at 149 VNPs was analyzed among the three passages. However, MAF decreased and mixed genotypes became unequivocally fixed to vaccine type in 23 vaccine-specific positions in higher passages of PPS. Plaque-purified Suduvax appeared to adapt to better replication during in vitro cell culture. Further studies with other vaccine strains and in vivo studies will help to understand the evolution of the VZV vaccine.
Asunto(s)
Vacuna contra la Varicela/genética , Herpesvirus Humano 3/crecimiento & desarrollo , Herpesvirus Humano 3/genética , Polimorfismo Genético , Cultivo de Virus , Línea Celular , ADN Viral/genética , Genoma Viral , Herpesvirus Humano 3/aislamiento & purificación , Humanos , Mutación , Análisis de Secuencia de ADN , Ensayo de Placa ViralRESUMEN
The directed molecular evolution of enzyme in vitro can not only improve the efficiency of evolution, but also evolve enzyme according to the investigator's desire. This review summed up the feasible methods of this novel technique.