`
`PCT/NL02/00390
`
`45
`
`further by examining the expression of genes in the Vicinity of the elements in
`
`the human genome, using public databases of DNA microarray
`
`(http://arrays.rockefeller.edu/xenopus/links.html) and SAGE (Serial Analysis
`
`of Gene Expression; http://bioinf0.amc.uva.nl/HTM—bin/index.cgi) data.
`
`10
`
`15
`
`20
`
`Protocol: STAR elements are tested in the pSDH plasmid, and SINC elements
`
`in the p88 plasmid. Three cell lines are transfected using standard protocols:
`
`the human U-2 OS osteosarcoma cell line (Heldin et a1., 1986), the Vero cell
`
`line from African green monkey kidney (Simizu et a1., 1967), and the CHO cell
`
`line from Chinese hamster ovary (Kao and Puck, 1968). Elements able to
`
`function in all three cell types are catagorized as promiscuous. Those only
`
`displaying activity in one or two of the cell-lines are catagorized as restricted
`
`in their cell—type functionality.
`
`Promoter specficity: STAR and SINC elements are currently selected and
`
`tested in the context of function with two promoters, the entire
`
`cytomegalovirus (CMV) promoter or the Tetracycline Response Element and
`
`minimal CMV promoter (in combination with the tTA transcriptional
`
`activator). To assess promoter specificity, STAR and SINC function are tested
`
`with other commonly used viral promoters, namely the simian Virus type 40
`
`(SV40) early and late promoters, the adenoviral ElA and major late
`
`promoters, and the Rous sarcoma virus (RSV) long terminal repeat (Doll et a1.,
`
`1996; Smith et a1., 2000; Weaver and Kadan, 2000; Xu et a1., 1995). Each of
`
`these promoters are cloned separately into the pSelect and p88 plasmids by
`
`standard techniques (Sambrook et a1., 1989) along with STAR or SINC
`
`25
`
`elements, respectively. The resulting plasmids are transfected into the U-2 OS
`
`cell line and assayed for reporter gene expression, as described above. The
`
`ability of SINC elements to silence these promoters, or STAR elements to
`
`protect against silencing, is determined by comparison with plasmids lacking
`
`STAR or SINC elements.
`
`30
`
`
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