The methylation-free status of a housekeeping transgene is lost at high copy number.
Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.
LGME-CNRS, Strasbourg, France.
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Mehtali
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LeMeur
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Lathe
D000818:Animals; D015139:Blotting, Southern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005796:Genes; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics*; D008745:Methylation; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D004274:DNA, Recombinant; D006903:Hydroxymethylglutaryl CoA Reductases; D015500:Chloramphenicol O-Acetyltransferase