Figure 1
From: Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c
Bioluminescent imaging allows allelic readout of imprinted Cdkn1c expression. (a) Schematic illustrating the mouse Cdkn1c locus and bioluminescent reporter strategy (left), and its application in vitro and in vivo55 (right). Allelic gene expression and DMR methylation status at early development (closed circles = methylated CpGs, open circles = unmethylated CpGs) are shown for the locus. The sDMR is unmethylated on both alleles until E6.524. The reporter construct, comprising firefly luciferase (Fluc), β–galactosidase (lacZ) and T2A sites, is inserted before the Cdkn1c stop codon in exon 3. Primers to distinguish expression of WT Cdkn1c and the reporter allele (Cdkn1c-Fluc) are shown by open and blue arrows respectively. Reporter mESCs were previously generated and used to create an in vivo model which exhibited proper imprinting of the reporter construct across generations (bioluminescent signal (blue) only present with maternal inheritance)55. Animals inheriting the reporter maternally or paternally can be used to derive corresponding reporter cells. Partially created with BioRender.com. (b) Schematic (left) and bioluminescent images (right, 6-well plate (wp)) of two mESC clones in which the reporter construct is inserted at either the maternal (clone B07) or paternal (clone H05) Cdkn1c allele. Since Cdkn1c is maternally expressed and paternally silenced, the reporter construct should be expressed in B07 cells and repressed in H05 cells, with the WT Cdkn1c allele exhibiting the reciprocal pattern. (c) Quantification of bioluminescent signal from B07, H05 and WT (no reporter) mESCs (n = 3; bars indicate mean; error bars represent standard deviation (SD); One-Way ANOVA p < 0.0001; results of Holm-Å Ãdák’s multiple comparisons follow-up test are shown: ****padj < 0.0001, **padj  = 0.0012). (d) Relative expression of Cdkn1c-Fluc (left) and WT Cdkn1c (right) alleles in B07 and H05 mESCs measured by RT-qPCR (n = 3; bars indicate geometric mean; error bars represent geometric SD; ****p < 0.0001, unpaired two-tailed t-tests on delta-Ct values). Primer positions to distinguish WT and reporter Cdkn1c alleles are illustrated in (a). (e) Western blot detection of CDKN1C protein in B07 and H05 mESCs using Lamin B1 as a loading control. The uncropped, unprocessed blot is available in the Supplementary Information file S1. (f) BLI of defined numbers of B07 and H05 mESCs in 48-well plates, showing representative images (left) and quantification (right). Quantification is relative to 4 × 104 B07 mESCs for each dilution series (n = 4 independent dilution series; simple linear regression; multiple two-sided t-tests with Holm-Å Ãdák’s correction comparing B07 to H05 for each dilution (****padj < 0.0001, ***padj < 0.001, **padj < 0.01).
