Abstract

The 4th International Workshop on Advanced Genomics

Kyoto International Conference Hall, Kyoto, Japan

November 13, 2001

P-41

Poster presentation

Analyses of transgenic mice bearing the differentially methylated CpG island of the mouse imprinted gene Impact

Kohji Okamura¹, Yoichi Yamada^1,3, Yuriko Hagiwara-Takeuchi¹, Yoshiyuki Sakaki^1,2, and Takashi Ito³
¹Hum. Genome Ctr., Inst. Med. Sci., Univ. Tokyo, Japan, ²Hum. Genome Res. Gr., RIKEN Genomic Sci. Ctr., Japan, ³Div. Genome Biol., Cancer Res. Inst., Kanazawa Univ., Japan

The mouse Impact is a paternally expressed gene encoding an evolutionarily conserved protein of unknown function. But its human homolog IMPACT is expressed biallelically. Genome sequence comparison revealed that the two genes share a well-conserved exon-intron organization but bear significantly different CpG islands. The mouse island lies in the first intron and contains characteristic tandem repeats. Furthermore, this island serves as a differentially methylated region (DMR) consisting of a hypermethylated maternal allele and an unmethylated paternal allele. Intriguingly, this intronic island is missing from the nonimprinted human IMPACT, whose sole CpG island spans the first exon, lacks any apparent repeats, and escapes methylation on both chromosomes. In this study we made transgenic mice bearing the mouse Impact CpG island excessively and examined them in order to discuss possibility of involvement in establishment and maintenance of the imprinting. 1.6-kb genomic DNA fragment containing the island was injected into fertilized eggs of C57BL/6J (B6). We obtained eight transgenic founder mice and six of them had their progeny. Transgene positive B6 were mated with another inbred strain, JF1/Msf (JF), derived from Mus musculus molossinus. The reciprocal F₁ hybrid mice, (JF × B6) F₁ and (B6 × JF) F₁, were used for allele-specific expression and methylation analyses. We looked into the six lines so far, using restriction fragment length polymorphism (RFLP) analysis and HapII-McrBC PCR assay. These results show that the paternal expression and the hypermethylation of maternal allele are not disturbed regardless of the numbers of the transgene copies and their methylation status. Now we are investigating the effect on genomic DNA methylation more precisely.