297. Development of HVJ-E High Throughput Gene Function Screening System: Screening of Endothelial Cell Growth Factors

Abstract

High Throughput screening system (HTS) is a powerful tool that can screen out gene function from pool of genes. Here, we developed a new HTS system using Hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector, which can be constructed by incorporating plasmid DNA into inactivated HVJ particles. After virus genome of HVJ is inactivated and broken down, the space inside of viral envelope is free to enclose variety of molecules such as pDNA, oligonucleotide, siRNA, antibody drug etc. In addition to the package ability, HVJ-E vector has wide range of target cells, both animal cells (especially primary and ES cells) and various tissues, by cell fusion activity. In this study, we screened endothelial growth genes to find out a new angiogenic factor. Human heart cDNA library (Gibco) was infused into HVJ-E vector and transfected to human aortic endothelial cells (HAEC; Clontech) that had been seeded into 96 well plates. For transtection, plasmid DNAs infused HVJ-E vectors were added to the medium of HAEC, and the plate was centrifuged for 1 hour. The plasmid DNA transfected HAEC were cultured in lower serum concentration medium (1% FBS) to reduce cell viability for seven days. We measured the HAEC viability by MTS assay. From the well of top score, DNA of cells were collected and transformed into E. coli to efficiently collect the candidate plasmids, because only plasmid DNAs, not cellular genomic DNAs, are formed in single clones (1st screening step of this method). We got couple thousands candidate genes from this step. All of the genes were sequenced, and homologies were searched by nucleotide-nucleotide BLAST (blastn). Based on the results of homologies, we have sorted genes that are secretion, signal transduction, transcription or unknown factors. The couple thousands of genes was narrow down to couple hundreds in this step. MTS assay were carried out again (2nd screening step of this method). Finally, we have isolated several novel genes that strongly promote HAEC growth. Further evaluation of newly found novel genes showed that they highly give rise to c-fos and VEGF promoter activation in endothelial cell. Overall, only two rounds of screening, 1st and 2nd screening, are sufficient to obtain target gene(s). In addition, all screened genes are retained in a plasmid form throughout whole screening steps, so that it saves much time and reduces chances of mutation during cloning procedures. HTS using HVJ-E vector will be a useful and powerful tool for screening the function of a lot of genes in four to six weeks Figure 1.