Title

Expression and methylation patterns of imprinted genes in deceased newborn clones and surviving adult clones in cattle

Date of Completion

January 2006

Keywords

Biology, Molecular|Biology, Genetics|Agriculture, Animal Culture and Nutrition|Biology, Veterinary Science

Degree

Ph.D.

Abstract

The objective of this research is to investigate the expression and methylation patterns of imprinted genes of somatic cell bovine clones, particularly the gene for Insulin-like growth factor receptor 2 (Igf2r). ^ Six candidate imprinted genes were isolated and identified in bovine, including Igf2r intron 2, 3'UTR of Igf2, H19, Snrpn, Peg1 and p57kip2. Maternal allelic expression of Igf2r was examined using single nucleotide polymorphism (SNP) in 9 organs: bladder, brain, heart, kidney, liver, lung, placenta, spleen and thymus. Random preferential expression of either allele of Igf2r in the placentas was found in clones, suggesting that cloning erased and re-established the epigenetic marks imprinted on the Igf2r gene. In contrast, the Igf2r expression pattern of the donor cells was faithfully preserved in all other clone tissues examined, except for the brain (biallelic expression). Further methylation patterns were detected in exon 1 and intron 2 of Igf2r. Varied methylation patterns among clones were observed in intron 2 of placenta and liver, even though clones were produced by the same genetic donor. The mRNA expression level of the bovine Igf2, Igf2r and H19 genes was also quantitated by Real time PCR in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen and thymus) of somatic cell cloned calves that died shortly after birth, in three tissues (skin, muscle and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. Deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs. Large variations in the expression levels of imprinted genes were also seen among these clones. In surviving adult clones, however, the expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data suggest that nuclear transfer can cause disruptions of expression of imprinted genes in bovine clones, possibly due to incomplete reprogramming of donor cell nuclei, and these abnormalities may contribute to the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only physically healthy but also relatively normal at the molecular level. ^