758. A Multipurpose Vector System for the Simultaneous Screening of Libraries in Bacteria, Insect and Mammalian Cells and Expression In Vivo

Abstract

Top of pageAbstract The post-genomic era necessitates new tools to decode the vast nucleotide sequence data into biologically relevant knowledge. This problem has created a new field in molecular biology and genetics called functional genomics, which addresses the functions of genes and gene products at the level of whole organisms. Viral vector systems have been explored as one solution to solve these problems. We have constructed a new baculovirus expression vector system (BEVS) based tetra-promoter vector (pBVboostFG) that enables screening of gene or cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, insect and bacterial cells. In addition, it is also suitable for direct use in vivo in gene therapy. Recombinant virus preparation is based on an improved mini Tn7 transpositional system that allows straightforward and fast production of baculoviruses with high diversity and negligible background. Genes or cDNAs were cloned into pBVboostFG using the bacteriophage lambda's recombination system and production of 20 different proteins was studied in bacteria, insect and mammalian cells. As an example of using the vector for library screening, a chromophoric Thr65-Tyr-Gly67-stretch of EGFP was destroyed and subsequently restored by PCR and the EGFP library was screened for the color variants. All studied proteins were efficiently produced using pBVboostFG system and with different expression hosts. In addition, three different fluorescent protein variants were screened out from the generated chromophore library. Finally, the BVboostFG vector was used without any additional subcloning steps to efficiently transduce choroid plexus epithelial cells in rat brain in vivo. The pBVboostFG system enables easy and efficient production of desired proteins or screening of genome-wide libraries in multiple cell types with one-step cloning. This makes it an efficient new platform technology for functional genomics and gene therapy.