The Analysis On The Genetic Stability Of Foreign Gene Expression And Security Of The Transgenic Potato System Expressing Human Interleukin-12

Abstract:Objective:To investigate the genetic stability of foreign gene expression and security of the transgenic potato expression system established to express hIL-12 by our research group.Methods:(1)The transgenic potatoes from three to four generations were planted and the hIL-12 gene transfected in potatoes was detected by polymerase chain reaction(PCR).(2)The expressions of hlL-12 gene of transgenic potatoes in three generations were tested by ELISA and Western blot.(3)EL1SA was also used to assess IFN-γsecretion of Peripheral blood mono-nuclear cells(PBMC) induced by hIL-12 in the three generations transgenic potatoes.The anti-tumor function of hIL-12 was detected by MTT method.(4)PCR was employed to amplify hIL-12 gene from earthworms which lived in the soil planting transgenic potatoes.ELISA was applied to assess hIL-12 level of earthworms and soil planting transgenic potatoes.(5)Transgenic potatoes,wild potatoes and normal diet were used respectively to feed rats for 14 days, and the several major organs of the rats were observed by histopathological exmination. Results:(1)hIL-12 gene was found in the transgenic potatoes of all three generations, but not in the wild potatoes.(2)Recombinant hIL-12(rhIL-12) was found in the transgenJc potatoes of three generations showed as a 35kD protein band,but not in the wild type.We found that hIL-12 level in the leaves and tubers of the transgenic potato plants in all three generations was obviously different from wild type group(P＜0.01). (3)Similar to the commercial rhIL-12,the purified hIL-12 was able to induce PBMC to secrete IFN-γin vitro and enhance cytotoxicity against A549.(4)The hIL-12 gene was not found in the earthworms from the soil samples of planting transgenic potatoes.The hIL-12 protein levels of the soil and the earthworms were not different from the wild type group.(5)The pathological condition was not found in the several major organs of the rats which were fed with transgenic potatoes,wild potatoes,and normal diet. Conclusion:(1)The hIL-12 transgenic potatoes established by our research group can stably inherit the hIL-12 gene to next generation.The hIL-12 gene in the next generation of transgenic potatoes can steadily express the hIL-12 which has the biological activity. (2)The hIL-12 gene in the transgenic potatoes does not drifted into the soil and the organism living in the soil.The transgenic potatoes are not poisonous.