Construction Of A Recombinant Human FGF1 Mammary Gland-specific Expression Vector And Identification Of Its Validity

Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors and are key players in the processes of cell proliferation, survival, differentiation and migration. In humans, 23 members of the FGF family have been identified, FGF1-FGF23. FGF1, also known as acidic fibroblast growth factor (aFGF), exhibits broad mitogenic activity in various cell types, including fibroblasts, myoblasts, endothelial cells, chondrocytes, osteoblasts, glial cells, astrocytes and smooth muscle cell. In addition to regulating mitogenic activity in embryonic development, morphogenesis, angiogenesis, wound healing and tissue repair, FGF1 exerts endocrine hormone-like activities including cardio-protection, vasodilation and neural regulation.Due to its wide range of biological activities acting on a variety of promising targets, FGF1 has excellent prospects as a therapeutic for a number of diseases. Therefore, an optimized expression system will be necessary to produce the quantity needed for clinical applications. The concept of using animal mammary glands as bioreactors to produce recombinant pharmaceutical proteins has been widely accepted as a commercially viable method because transgenic animal mammary gland bioreactors can produce recombinant proteins with appropriate post-translational modifications; additionally, milk provides a safe, abundant, and easily obtainable source of raw material for the purification of expressed recombinant protein.Although mammary gland bioreactors have great commercial value, most foreign genes have not been successfully expressed at sufficiently high levels to make this method commercially viable. The most important reason for the low level of transgenic expression is that transgenes integrate into the genome randomly, resulting in the inhibition of transgene expression by the surrounding chromatin structures. Therefore, the key to improving the expression of foreign genes is to reduce the adverse effects of the position of integration. Additionally, selectable marker genes are indispensable to ensure efficient genetic modification with transgenes. However, following the selection of stable cell lines, these marker genes lead to problems in the generation of transgenic animals. The Cre/loxP system has been developed as a powerful tool for gene deletion and inversion. Cre-mediated deletion has been successfully applied to achieve marker removal to produce transgenic plants. High quality and low expense methods of producing transgenic animals are highly regarded by investigators. Cre-mediated cassette exchange (CMCE) is an economical method to replace the first gene expressed in a favorable locus with the gene of interest to generate a bioreactor without requiring the selection of favorable loci.In this study, we constructed an FGF1 mammary gland-specific expression vector (pBC1-PIE-FGF1) containing the elements to screen for favorable loci, delete marker genes and induce another gene of interest. FGF1, driven by goatβ-casein promoter, was designed for specific expression in mammary gland. Enhanced green fluorescent protein (EGFP) was used to select a favorable location for transgene expression. The expression levels of transgenes at different chromosomal locations can be evaluated by detecting the fluorescence intensity of EGFP. A high level of EGFP expression indicated favorable locations where the vector integrated into the genome. Various lox sites were also inserted into this vector. Two loxP sites with the same orientation were introduced on either side of the selectable marker genes (EGFP and the puromycin resistance gene, Puror) and were used to excise these marker genes from the genome after they fulfilled their purposes. The heterospecific lox sites lox66 and lox2272 flanking FGF1 were selected for Cre-mediated recombination. Using the Cre recombinase in combination with the heterospecific lox sites allowed for the rapid induction of other transgenes into defined loci. The constructed vector was validated in MCF-7 cells by examining the expression and mitogenic activity of FGF1 as well as Cre-mediated deletion and inversion. The results show that this vector is suitable for further use in the generation of transgenic mammary gland bioreactors.