Safety Assessment Of Intratesticular Injection And Biological Safety Assessment

Lentivirus is a retrovirus containing polymerase dependent on RNA which is known as reverse transcriptase, lentivirus use viral RNA as a template to synthesize cDNA by reverse transcriptase in host cells, and then use this cDNA as a template to synthesize double-stranded DNA, integrate into the host cell genome by the integration enzymes in the virus. Lentiviral vector built on lentiviral has high transfection efficiency and causes little immune response, etcTesticular injection method is the use of testicular microinjection of exogenous DNA injected directly into the male animals, the exogenous DNA into the testis and sperm interactions, the transfection of sperm cells. The method is simple, whether it is safe, effective, needs to be further verified.This trial looked at the animal’s own health security through the acquisition of testis after injection of the experimental cattle blood and semen PCR assay to determine whether the integration of exogenous DNA and sperm in the testis spread to the body tissues; experimental cattle semenconventional microscopic examination for detection and sperm quality electronic map to study the impact on sperm in the testicular injection of bovine testicular tissue and slow integration of the virus and sperm.The research mainly consists of two parts:1.Testicular injection of Lentivirus on the reproductive performance of bullsWe using testicular multi-point injection, then multiple semen to detection the quality of semen including the main indicators of the density, vitality and appearance and color.And observe the sperm morphology and density of Semen smears under the electron microscope of400times.Results show that:experimental group of cattle semen density, vitality, slightly lower than the control group cattle semen. Under the electron microscope of400times, Sperm density of the experimental group lower than the control group, but Sperm morphology is normal and abnormal sperm rate is low.2. The exogenous DNA detection in blood and semenIn this experiment, routine blood analysis and the exogenous gene PCR amplification to ensure the lentiviral vector gene transfer spread to other tissues in the host body or not. Our laboratory PCR sensitivity can make10copy number amplificating external source BSD gene and PCR products were1%agarose gel electrophoresis.Results show that:the routine testing of blood were not significantly different of the experimental group and control group (p>0.05)3. Testicular injection of Lentivirus on the blood physiological and biochemical indexesIn this experiment, we use preliminary statistics of the indicators on Microsoft Office Excel2007to data blood testing and make single-factor analysis of variance (ANOVA) by SAS8.0software.Results show that:We are not detected the sequence of exogenous BSD in the blood and in the semen.These results indicate that:testicular injection of lentiviral vectors will not produce damage for cattle breeding and health and will not proliferate to infect other organizations in the host body.This study could provide a support for the safety of lentiviral vector-mediated RNAi technology to prepare genetically engineered bovine. In addition, this study could also provide a reference on the use of testicular injection for genetically modified cattle.