GENE SILENCING BY DNA METHYLATION AND DUAL INHERITANCE IN CHINESE-HAMSTER OVARY CELLS

Chinese hamster ovary (CHO) cells strain D422, which has one copy of t he adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphat e. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine . Colonies were isolated and shown to be reactivated to APRT(+) by 5-a za-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of indepe ndent origin and subjected to bisulphite treatment. This deaminates cy tosine to uracil in single-stranded DNA but does not deaminate 5-methy l cytosine. PCR, cloning and sequencing revealed the methylation patte rn of CpG doublets in the promoter region of the APRT-gene, whereas th e active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT(+) gene, could also be silenced by the same proced ure but at a lower frequency. The availability of the 5-methyl dCTP-in duced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulpho nate, makes it possible to follow concomitantly the inheritance of act ive, mutant or silenced gene copies. This analysis demonstrates ''dual inheritance'' at the APRT locus in CHO cells.