Expression Of Human Transferrin In Transgenic Animals

Transferrin (TF) is an iron-binding, monomeric glycoprotein. It is an important factor involved in various cellular processes and plays complex physiological roles related to cell function, differentiation and proliferation. Conceivably, production of TF through transgenic approaches holds great potentials in bio-pharmaceutical industry.In this study, We have analyzed the capabilities of several regulatory elements in regulating the expression of human TF in diverse tissues of animal models. First, the enhancer and promotor of rabbit transferrin were cloned by PCR and ligated with human transferrin minigene (including TF cDNA and intron I). Meanwhile, human transferrin minigene was ligated with goat beta lactoglobulin promotor as control. Then, these expression elements were cloned into lentivirus vector. To investigate the expression of human transferrin, these plasmids were transfected into HC-11, BRL-3A cell lines and mouse or goat mammary gland, respectively. The results showed that human transferrin could be expressed in these cell lines and mouse or goat mammary gland following transfection, and its expression level could be improved dramatically when it was driven by rabbit enhancer and promotor as compared with goat beta lactoglobulin promotor. Transgenic mice were generated by either microinjection or lentivirus infection. The expression level of human transferrin was analyzed and the efficiencies of two different preparation methods were studied. The experimental data confirmed that human transferrin could be expressed at the highest level in the serum when it was regulated by the element including one rabbit enhancer and promotor. The expression level reached as high as 913.6μg/mL and 697.3μg/mL in serum and milk of the transgenic mice, respectively. The results also suggested that the transgenes could be not only integrated into the host chromosomes more efficiently (the integration rate was improved three times), but also expressed at higher level when the transgenic mice were prepared with lentivirus infection other than microinjection. However, the expression profiles of human transferrin in different organs of transgenic mice were not changed when the mice were prepared with different methods. Human transferrin was mainly expressed in liver and mammary gland of transgenic mice, and the expression levels in other organs such as heart, kidney and so forth, were very low.Meanwhile, the integration and expression of human transferrin in transgenic fetal bovine, which was prepared with in vitro transfection and somatic nuclear transfer, were analyzed. The data indicated that foreign gene was integrated into bovine chromosome with only one copy, and the integration site was located in the downstream of one funtional gene. And the expression of human transferrin was detected in liver of fetal bovine.In addition, two RNAi fragments targeting mouse beta casein gene and three RNAi fragments targeting whey acidic protein gene were synthesized and cloned into expression vectors. These vectors were injected into the mammary gland of transgenic mice integrated with human transferrin gene, and the relative quantity of beta casein and concentration of human transferrin were analyzed. The results showed that the concentration of endogenous milk protein was decreased and the expression levels of human transferrin were improved in most of the transgenic mice. Then, transgenic mice integrated with RNAi fragments were prepared and the data indicated that the relative abundance of beta casein in the milk of one transgenic mouse reached only 70% of normal mice.In this paper, the expression levels of human transferrin in different organs of transgenic animals were analyzed, and the data revealed that human transferrin could be expressed effectively in the serum and milk of transgenic mice when proper regulatory elements were utilized. Meanwhile, it was found that the expression of transgenes could be improved when the endogenous milk proteins were inhibited. These data should provide valuable information and offer a new idea to promote the study of transgenic mammary gland bioreactor.