Optimization Of Chicken Oviduct-Specific Expression Vectors For Transient Expression Of Recombinant Proteins

Many gene expression systems have been established for production of large quantities of biologically active proteins for clinical and scientific research use, trans-genic animal bioreactors in particular. Among animal bioreactors, transgenic animal mammary gland and avian oviduct are expected to be excellent systems for production of recombinant proteins. Transgenic avian oviduct bioreactors have advantages of high expression levels and low cost. However, research on avian transgenesis is greatly lagged behind other animals due to the complexity of avian physiology.To develop chicken bioreactors for transient expression of recombinant proteins, oviduct-specific expression vectors have been constructed using the 3.0 kb 5′- and 3′- regulatory regions of chicken ovalbumin (ov) gene, and have been shown to express relatively high levels of recombinant enzymes using the genes of interest of human lysozyme and human tissue kallikrein (hK1). In this study, we optimized the structure of the previously constructed vector pOV3K by restriction digestion or PCR amplifica-tion to shorten the 5′-regulatory region to 2.1, 1.7 and 1.1 kb, and the modified vectors were named pOV4K, pOV5K and pOV6K, respectively. The new vectors and the con-trol vector pOV3K were injected into laying hens via wing vein route following mixing with 25 kDa polyethyleneimine (PEI). The eggs were collected and the enzymatic ac-tivity in egg white was detected. Among the four vectors tested, pOV5K with the 1.7 kb 5′-regulatory region showed an expression level similar to that of pOV3K. To further improve the transfection efficiency and expression levels, the expression cassette con-sisting of the 1.7 kb 5′- regulatory region of ov gene, hK1 cDNA and bovine growth hormone gene poly (A) was cut from the pOV5K vector and subcloned into the pITRLRep vector containing the inversed terminal region (ITR) and Rep gene of avian...