Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation.
Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.
Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, United States of America.
Julia V
JV
Kirov
Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, United States of America.
Michael
M
Adkisson
Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, United States of America.
A J
AJ
Nava
Mouse Biology Program, University of California Davis, Davis, CA, United States of America.
Andreana
A
Cipollone
Mouse Biology Program, University of California Davis, Davis, CA, United States of America.
Brandon
B
Willis
Mouse Biology Program, University of California Davis, Davis, CA, United States of America.
Eric K
EK
Engelhard
Mouse Biology Program, University of California Davis, Davis, CA, United States of America.
K C Kent
KC
Lloyd
Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, United States of America.
Pieter
P
de Jong
Children's Hospital Oakland Research Institute (CHORI), Oakland, CA, United States of America.
David B
DB
West
D000818:Animals; D018899:CpG Islands; D019175:DNA Methylation; D020869:Gene Expression Profiling / Q000379:methods; D020868:Gene Silencing; D017930:Genes, Reporter; D007763:Lac Operon; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis*
D012333:RNA, Messenger