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J Biol Chem, Vol. 274, Issue 34, 24232-24240, August 20, 1999
From the Institute of Genetics, University of Cologne,
D-50931 Koeln, Germany
Integration of foreign DNA into an established
host genome can lead to changes in methylation in both the inserted DNA
and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the
integration site or inserted DNA can affect de novo
methylation. Homologous recombination was used to integrate foreign DNA
into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic
stem (ES) cells. Two plasmids were chosen for integration; one
contained the adenovirus type 2 E2AL promoter upstream of the
luciferase reporter gene, and the second carried the early SV40
promoter. The methylation patterns were analyzed using
HpaII and MspI restriction endonucleases for
both homologously recombined and randomly integrated foreign DNA in the
ES cell clones.
Upon homologous reinsertion of the BLK gene into the genome of mouse ES
cells, methylation patterns in this gene were reestablished. In DNA
segments adjoined to the BLK gene, the de novo patterns of
DNA methylation depended on the viral sequences in these clones and on
the locations of the inserts, i.e. on whether the
insertions resulted from homologously recombined or randomly integrated
foreign DNA. In homologously recombined DNA, sequences carrying the
adenovirus type 2 promoter were heavily methylated, and those with an
SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the
promoter and enhancer element present in the construct. These results
indicate that modes and sites of integration as well as the inserted
nucleotide sequence, possibly promoter strength, are factors affecting
de novo methylation.
Factors Affecting de Novo Methylation of Foreign DNA
in Mouse Embryonic Stem Cells
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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