Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2015 Aug 15

PMID 26275310

Citations 2

Authors

Julia V Kirov

Michael Adkisson

A J Nava

Andreana Cipollone

Brandon Willis

Eric K Engelhard

K C Kent Lloyd

Pieter de Jong

David B West

Affiliations

Soon will be listed here.

Abstract

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.

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References

Soriano P . Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21(1):70-1. DOI: 10.1038/5007. View

Hyvarinen J, Hassinen I, Sormunen R, Maki J, Kivirikko K, Koivunen P . Hearts of hypoxia-inducible factor prolyl 4-hydroxylase-2 hypomorphic mice show protection against acute ischemia-reperfusion injury. J Biol Chem. 2010; 285(18):13646-57. PMC: 2859527. DOI: 10.1074/jbc.M109.084855. View

Ryder E, Gleeson D, Sethi D, Vyas S, Miklejewska E, Dalvi P . Molecular characterization of mutant mouse strains generated from the EUCOMM/KOMP-CSD ES cell resource. Mamm Genome. 2013; 24(7-8):286-94. PMC: 3745610. DOI: 10.1007/s00335-013-9467-x. View

Jjingo D, Conley A, Yi S, Lunyak V, Jordan I . On the presence and role of human gene-body DNA methylation. Oncotarget. 2012; 3(4):462-74. PMC: 3380580. DOI: 10.18632/oncotarget.497. View

Whitelaw E, Martin D . Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat Genet. 2001; 27(4):361-5. DOI: 10.1038/86850. View

Scacheri P, Crabtree J, Novotny E, Chen A, Edgemon K, Marx S . Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice. Genesis. 2001; 30(4):259-63. DOI: 10.1002/gene.1072. View

Hellman A, Chess A . Gene body-specific methylation on the active X chromosome. Science. 2007; 315(5815):1141-3. DOI: 10.1126/science.1136352. View

Jones P, Baylin S . The epigenomics of cancer. Cell. 2007; 128(4):683-92. PMC: 3894624. DOI: 10.1016/j.cell.2007.01.029. View

Tajima S, Shinohara K, Fukumoto M, Zaitsu R, Miyagawa J, Hino S . Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells. J Biochem. 2006; 139(4):705-14. DOI: 10.1093/jb/mvj075. View

10.

West D, Pasumarthi R, Baridon B, Djan E, Trainor A, Griffey S . A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines. Genome Res. 2015; 25(4):598-607. PMC: 4381530. DOI: 10.1101/gr.184184.114. View

11.

Birling M, Gofflot F, Warot X . Site-specific recombinases for manipulation of the mouse genome. Methods Mol Biol. 2009; 561:245-63. DOI: 10.1007/978-1-60327-019-9_16. View

12.

Zuniga A, Michos O, Spitz F, Haramis A, Panman L, Galli A . Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. Genes Dev. 2004; 18(13):1553-64. PMC: 443518. DOI: 10.1101/gad.299904. View

13.

Skarnes W, Rosen B, West A, Koutsourakis M, Bushell W, Iyer V . A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 2011; 474(7351):337-42. PMC: 3572410. DOI: 10.1038/nature10163. View

14.

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

15.

Krueger F, Andrews S . Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011; 27(11):1571-2. PMC: 3102221. DOI: 10.1093/bioinformatics/btr167. View

16.

Karolchik D, Baertsch R, Diekhans M, Furey T, Hinrichs A, Lu Y . The UCSC Genome Browser Database. Nucleic Acids Res. 2003; 31(1):51-4. PMC: 165576. DOI: 10.1093/nar/gkg129. View

17.

Olson E, Arnold H, Rigby P, Wold B . Know your neighbors: three phenotypes in null mutants of the myogenic bHLH gene MRF4. Cell. 1996; 85(1):1-4. DOI: 10.1016/s0092-8674(00)81073-9. View

18.

Masser D, Berg A, Freeman W . Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing. Epigenetics Chromatin. 2013; 6(1):33. PMC: 3907040. DOI: 10.1186/1756-8935-6-33. View

19.

Hadjantonakis A, Pirity M, Nagy A . Cre recombinase mediated alterations of the mouse genome using embryonic stem cells. Methods Mol Biol. 2008; 461:111-32. PMC: 2883169. DOI: 10.1007/978-1-60327-483-8_8. View

20.

Barski A, Cuddapah S, Cui K, Roh T, Schones D, Wang Z . High-resolution profiling of histone methylations in the human genome. Cell. 2007; 129(4):823-37. DOI: 10.1016/j.cell.2007.05.009. View