Real-time PCR analysis of candidate imprinted genes on mouse chromosome 11 shows balanced expression from the maternal and paternal chromosomes and strain-specific variation in expression levels

Abstract

Imprinted genes are monoallelically expressed from either the maternal or paternal genome. Because cancer develops through genetic and epigenetic alterations, imprinted genes affect tumorigenesis depending on which parental allele undergoes alteration. We have shown previously in a mouse model of neurofibromatosis type 1 (NF1) that inheriting mutant alleles of Nf1 and Trp53 on chromosome 11 from the mother or father dramatically changes the tumor spectrum of mutant progeny, likely due to alteration in an imprinted gene(s) linked to Nf1 and Trp53. In order to identify imprinted genes on chromosome 11 that are responsible for differences in susceptibility, we tested candidate imprinted genes predicted by a bioinformatics approach and an experimental approach. We have tested 30 candidate genes (Havcr2, Camk2b, Ccdc85a, Cntnap1, Ikzf1, 5730522E02Rik, Gria1, Zfp39, Sgcd, Jup, Nxph3, Spnb2, Asb3, Rasd1, Map2k3, Map2k4, Trp53, Serpinf1, Crk, Rasl10b, Itga3, Hoxb5, Cbx1, Pparbp, Igfbp4, Smarce1, Stat3, Atp6v0a1, Nbr1 and Meox1), two known imprinted genes (Grb10 and Impact) and Nf1, which has not been previously identified as an imprinted gene. Although we confirmed the imprinting of Grb10 and Impact, we found no other genes imprinted in the brain. We did, however, find strain-biased expression of Camk2b, 5730522E02Rik, Havcr2, Map2k3, Serpinf1, Rasl10b, Itga3, Asb3, Trp53, Nf1, Smarce1, Stat3, Cbx1, Pparbp and Cntnap1. These results suggest that the prediction of imprinted genes is complicated and must be individually validated. This manuscript includes supplementary data listing primer sequences for Taqman assays and Ct values for Taqman PCR.

Figure 1

Diagram of tested genes on mouse chromosome 11. Candidate genes were chosen that covered the length of chromosome 11. The centromere is at the top of the diagram and the telomere is at the bottom, with the Mb distance from the centromere indicated along the left side of the diagram.

Figure 2

Percentage B6 allele in maternal B6 cDNA, maternal B6 gDNA, paternal B6cDNA, and paternal B6 gDNA samples. Graphs show the average percentage of the B6 allele in samples with the standard deviation indicated as error bars. Grb10 (panel A) and Impact (panel B) are known imprinted genes and show very low expression of the B6 allele when it is inherited from the mother (red bar) and very high expression of the B6 allele when it is inherited from the father (blue bar), indicating that these genes are expressed from the paternal allele. The B6 allele makes up approximately 50% of the F1 genomic DNA when the B6 allele is inherited from the mother (red checked bar) or from the father (blue checked bar) as expected. Camk2b (panel C) is expressed less from the B6 allele, whereas Asb3 (panel D), Trp53 (panel E), and Nf1 (panel F) are expressed more from the B6 allele.