Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).</p><p><b>METHODS</b>A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189.</p><p><b>RESULT</b>The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG.</p><p><b>CONCLUSION</b>POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.</p>