Development Studies On Nuclear Transfer Embryos Transfected Human Lysozyme

The study of mammary gland bioreactor has become one of the hot spots in the biotechnology field, and the research on production of protein which has biological activity using mammary gland bioreactor have been Implemented widely and quickly. Human lysozyme, as a natural protein with function of anti-inflammatory and preservation,.have widely application in the life of people. However, human lysozyme protein extracted from human tissues and organs is limited and no more meet the current needs. In order to large production of human lysozyme, genetic engineering technologies, including transgenic somatic cell nuclear transfer, have been utilized in recent years.The purpose of this study was designed to make mammary gland bioreactor producing human lysozyme by using transgenic somatic cell nuclear transfer. Following results have been obtained:1. Bovine fibroblast cells were isolated by attaching tissue explants from ear skin of a bovine. The plasmid pEBH containing gene of hLYZ and two selecting marks(the EGFPand Neor gene) which expresss pecifically in mammary gland was transfected by lipofectmine 2000 into forth passages bovine fibroblast. A small part of the positive cells were analyzed by PCR and RT-PCR for gene integration. The both detections demonstrated that the foreign gene was integrated into the genome and could be expressed. The results indicated that the isolated bovine fibroblast cells might be competent for transgenic somatic cell cloning.2. To study the effect of transgenic transfection on the development potential of reconstructed embryos. Transgenic donor cells seclected by G418 were used as donor cells. No significant difference were found in fusion and cleavage rates between transgene transfected and non-transfected group. But the blastocyst development rate is lower in transfected group.(P<0.05). It indicated that transgene transfection has negative effect on transgenic reconstructed embryos.3. To improve the effeciency of transgenic somatic nuclear transfer, the effect of selection methods of transgenic donor cells on the production of transgenic reconstructed embryos was studied. We found that the blastocyst rate of reconstructed embryos was highest when transgene transfected cells with green fluorescent were selected and used as nuclear donor cell after one week of transfection, however, the positive blastocyst rate was only 75.8%. Although impaired development of cloned embryos was observed in combination of G418 and fluorescent selection, the positive blastocyst rate was as high as 100%. Single G418 selection was not a good method both in term of developmental competence and transgenic positive of reconstructed embryos (P<0.05).4. The time of green fluorescent expression in reconstructed embryos has obviously effect on the following development of transgenic cloned embryos. Reconstructed embryos expressing green fluorescent at 8-16 cells have a highest rate of blastocyst development.