Fig 1.
Schematic of Experimental Approach.
A cartoon of the knockout first allele is shown with exons indicated by gray blocks numbered 1–3. The targeted exon in this schematic is #2, with the targeting vector replacing that critical exon with an identical exon flanked with LoxP sites. Proximal to the critical exon are placed in tandem a LacZ reporter gene driven by the targeted gene promoter, followed by transcriptional stop and polyadenylation signal, and then followed by a heterologous promoter driving a neomycin resistance gene. The gene regions evaluated for expression by qRT-PCR and/or for methylation by sequencing bisulfite treated DNA are indicated in brackets.
Table 1.
qRT-PCR Primers and Probes.
Table 2.
CpG Island Primers.
Table 3.
LacZ primers used for Amplifying Reporter Gene Sequence.
Fig 2.
RQ mRNA of Targeted Genes and LacZ.
Silenced and Expressed group targeted gene expression, and LacZ reporter gene expression, are presented as Relative Quantity (2-ΔΔCt) values relative to native gene expression in Wild Type Control animals for each mutant gene and tissue. Each data point is the average of three biological replicates, with the qPCR done in triplicate.
Fig 3.
Methylation of CpG Islands in Targeted Genes.
Percent methylation of CpG islands (CGI) in Mutant tissues and in Wild Type controls are presented for the Silenced and Expressed groups. Although the differences were small for some CGIs the Cochran-Mantel-Haenszel test determined that each mutant CpG island was significantly different from the corresponding Wild Type Control island percent methylation.
Fig 4.
Targeted Gene Expression, Reporter Gene Expression and Promoter Methylation.
Individual mutant and tissue values were averaged across the Silenced group (n = 7) and the Expressed group (n = 18) and compared by t-test. * = p<0.01; ** = p<0.0001. Date are presented as means +/- standard errors. The Silenced group had significantly lower expression of the targeted gene and the LacZ reporter and had higher percent methylation compared with the Expressed group.
Fig 5.
Correlation between Promoter and LacZ Coding Sequence Methylation, and between Promoter Methylation and LacZ Expression in Silenced Mutants.
A significant positive correlation was observed between LacZ and CpG % methylation with a p < 0.013. Although the correlation between CpG% methylation and LacZ gene expression was negative as expected, this did not reach statistical significance.
Fig 6.
Methylation of CpGs in LacZ Coding Sequence.
% methylation of CpGs in the LacZ coding sequence is presented for each mutant and tissue, with Silenced mutants in red and Expressed mutants in blue. There was a significant overall difference in average percent methylation of LacZ coding sequence between the two groups (t-test; p < 0.001).