Abstract: Objective To study the mechanisms of the expression of exogenous gene injected via the mammary duct in rat mammary gland tissue.Methods Thirty-six SD rats were grouped according to the stages of mammary growth(9 weeks after birth,7,12 and 18 days of pregnancy,and 7 and 14 days post partum,6 rats in each stage).pCE-29 plasmid for highly efficient expression of green fluorescent protein(GFP)and in vivo jetPEITM were mixed and injected into the 5th pair of the mammary glands via the mammary ducts in each rat.The mammary glands were taken to prepare freezing slices 48 h after the injection and observed under fluorescence microscope.HE staining was performed for slices with GFP expression to locate the site of GFP expression by comparison of the images before and after staining.Paraffin sections of the mammary glands with HE staining were also obtained from normal rats in different stages of mammary development.Results After pCE-29 plasmid injection,GFP expression was observed in the mammary glands of 2 rats in pregnancy day 12 group and in postpartum day 7 group.The green spots shown by fluorescence microscopy were found in the mammary epithelial cells after image comparison.The gland cells undergoing cell division were more numerous in the two stages(pregnancy day 12 and postpartum day 7)than in other stages of mammary development.Conclusions In the two stages of pregnancy day 12 and postpartum day 7,exogenous genes injected via the mammary duct can be expressed in the mammary glands.In these stages,the stem cells in the mammary gland epithelia differentiate continuously and the mammary gland epithelial cells flourish under the regulation by hormones to allow better chance for exogenous gene acceptance in the context of active DNA replication in the gland cells.This finding provides important experimental evidence for further studies of mammary gland bioreactor in animals.