Construction of 700 human/mouse A9 monochromosomal hybrids and analysis ofimprinted genes on human chromosome 6

As an in vitro assay system for the identification of human imprinted genes , a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from n ormal human fibroblasts, has been previously generated by microcell-mediate d chromosome transfer (MMCT). To supplement this assay system, we construct ed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo sel ection marker. To validate the A9 hybrids, we screened them with chromosome -specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching pa ternal and maternal chromosome pairs of A9 hybrids were identified for chro mosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc fin ger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hy datidiform mole-associated and imprinted transcript), and the maternal-spec ific methylation of a CpG island within an imprinted domain on human chromo some 6q24, were maintained in A9 hybrids. For an example, we profiled the e xpression of expressed sequence tags (ESTs) and the methylation of CpG isla nds in the 300-kb imprinted domain around 6q24, which may be associated wit h cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.