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The demand of pharmaceutical proteins and continuously increasing worldwide, hence cost of production of these proteins is a key factor in the competitive market. Recombinant proteins have several benefits over proteins which are isolated from non-recombinant sources. There are several methods developed for expression of recombinant proteins in large scale. Among these mammalian cell culture based expression system is most used world wide followed by expression of recombinant proteins in live animal bioreactor. Although concept of generating therapeutic proteins in the mammalian cell cultures based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. One of the issues for lack of advancement in this field is exorbitant cost involved in downstream processing of the expressed proteins. This is largely due to lack of a cell culture system which may aid in high level expression of therapeutic protein followed by efficiently processing and secreation of the expressed protein in the culture. To achieve this, development of cheaper and less cumbersome procedures has become necessary. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modifications. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. The mammary gland has generally been considered the organ of choice to express valuable recombinant proteins because milk is easily collected in large volumes and is the best available bioreactor. However, large scale production of recombinant proteins is still an art in spite of increased qualitative and quantitative demand for these proteins. Foreign proteins are commonly reported to be produced in transgenic milk at rates of several grams per liter. Researchers are constantly challenged to improve and optimize the existing expression systems, and also to develop novel approaches to face the demands of producing the complex proteins. Although concept of generating therapeutic proteins in the mammalian cell culture based system existed for more than two decades, it is still less efficient for producing cost effective therapeutics. We have established bovine mammary epithelial stem cells (bMESCs) using various markers such as Nanog, Oct4, Sox9, CK14, CK18, CSN2, CSN3 etc. Antibacterial pharmaceutical proteins were screened using various bio-informatic tools and then selected proteins such human lipocalin-2 (LCN2) and bovine lactoferricin for the transfection in cow bMESCs. We used different mammary specific vector systems as career for the transfer of targeted genes in bMECs. However, transfection in mammary epithelial/ stem cells is little difficult as compare to other cells as these cells have secretory properties. We successfully expressed both pharmaceutical proteins in the cow mammary epithelial stem cells and established the cell lines. Therefore, development of protocols and establishment of cow mammary epithelial/stem cell lines can be used for various studies and as non-GMO bioreactor for the production of pharmaceutical value proteins.