| Background Cancer is a serious disease which threatens human’s health. Non-human primates are genetically and physically more similar to humans than other animal models,providing an important model for cancer study. P53 and P21 are popμlar tumor supressor gene,whose down regulation may lead to cancer in human.They can induce cell cycle arrest or apoptosis and contribute a lot to DNA damage repairment.Objective The down regμlation of P53 and P21 may induce cancer in animals. In the present study, we aim to silencing P53 and P21 of Rhesus Monkey gene on cell and embryo level.to make preparation for the cancer model foundation of Rhesus Monkey.Methods (1) The foundation of lentivirus mediated RNA interference plasmid carrier and Rhesus Monkey P53 and P21 gene silencing on cell level:Detect the abundance of P53 and P21 in COS-7 cells,Three small hairpin RNA (shRNA) sequences targeting rhesus monkey P53 gene were designed, analysed by bioinformatics and inserted into lentivirus-based gene silencing constructs FUGW-TDT. The plasmids of P53-RNAi P21-RNAi and control vector were transfected into the COS-7 cells respectively. P53 mRNA and proteins were detected by Real-time PCR and Western-blot. P53 and P21 gene expression was detected in COS-7 cells; Three P53 gene silence sequences and four P21 gene silence sequences were designed;(2) Lentivirus Package and P53 gene silencing on embryo level:Three gene silencing vectors were chosen to be packaged. Lentivirus was produced after the working vector FUGW-TDT and three package plasmids were cotransfected into 293T cells. The tilters were tested. Rhesus monkey females were selected for ovarian hyperstimulation twice, oocytes were collected via opration.20 MII oocytes were utilized for intraeytoplasmic sperm injecfion (ICSI).Lentivirus were microinjected into the perivitelline space of 8 oosperms. The other two were treated as blank control. The lentivirus infected embryo were cμltured in vitro until morμla stage,then they were observed by Fluorescence microscope.Resμlts (1) The foundation of lentivirus mediated RNA interference plasmid carrier and Rhesus Monkey P53 and P21 gene silencing on cell level:Four P53 gene-silencing sequences were screened that lied in 238-258 bp,681-701 bp,169-189 bp of the Rhesus monkey P21 mRNA, all of them lies in the ORF origin.48 hrs after transfection of the silencing constructs, P53 mRNA was suppressed by (87.17±4.03)%, (72.62±4.11)%, (76.22 ± 0.98)%; P53 protein was suppressed by (84.44±2.18)%, (71.04±1.18)%, (74.17±0.95)%; Four P21 gene-silencing sequences were screened that lied in 541-561 bp,542-562 bp,215-239 bp. and 624-648 bp of the rhesus monkey P21 mRNA, all of them lies in the ORF origin. P21 mRNA was suppressed by (91.82±3.21)%, (82.47±2.48)%,(81.31±2.69)%,(87.35±4.59)%.P21 protein was suppressed by (88.03±0.53)%, (79.78±0.42)%, (76.79±0.63)%, (85.58±0.61)%.(2) Lentivirus Package and P53 gene silencing on embryo level:The tilters were 2.51 ×109IU/ml,1.76×109IU/ml and 1.06×109 IU/ml It means lentivirus has successfμlly infected the embroys. Red fluorescence were seen in the 8 infected embryos while no fluorescence seen in the controls.Conclusions Rhesus monkey P53 and P21 gene were successfμlly silenced on cell level.Three effective silencing target sequences of Rhesus monkey P53 shRNA expression vector and four of P21 were screened; Preliminary test were done on embryo level. The packaged lentivirus were injected into embryos.Red fluorescence were seen in morμla stage, which lays important foundation for latter research.
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