249 EXPRESSION OF GROWTH-RELATED IMPRINTED GENES IN DECEASED NEWBORN AND SURVIVING CLONED PIGLETS

Abstract

Different from cloned mice and cattle, which are often born with large offspring syndrome (LOS), pigs produced through nuclear transfer have not been found to be overly large compared to their age-matched controls. In our study, significant differences (P < 0.05) were found in the body weight of both deceased newborn cloned piglets (n = 9) (1.2 ± 0.8 vs. 2.1 ± 0.2 kg) and cloned pigs at one month of age (n = 7) (6.1 ± 1.3 vs. 8.0 ± 0.8 kg), when compared to their controls (n = 5 for each age group). Because imprinted genes are important regulators of fetal growth and are subject to reprogramming during nuclear transfer, we aimed to determine the expression levels of both growth-enhancing and growth-inhibiting imprinted genes in these cloned pigs by real time quantitative reverse transcription polymerase chain reaction (RT-PCR). These genes include: Igf2 and Peg3 (growth-promoting), and Igf2r and Grb10 (growth-inhibiting). Tissues from six major organs were collected from clones and controls of both age groups. SYBR Green I was utilized as a reporter for the PCR reactions and 18S ribosomal RNA was used as the internal reference for quantifying the relative expression level of all genes. With the exception of Igf2, the other three imprinted genes were found to have statistically different levels of expression in certain organs of both groups of pigs when compared to their age-matched controls. However, no strong correlation was found between the levels of gene expression and the low-body-weight phenotype of the cloned pigs. Contrary to the notion that nuclear transfer can reduce the variation in the resulting animals due to their identical genetic make-up, larger variances of gene expression were found in clones of both groups, indicating the randomness of the nuclear reprogramming process. For all four imprinted genes studied, the coefficient of variation (CV) decreased greatly from newborn to one month old in both clones and controls, suggesting a more stringent regulation on imprinted gene expression after birth.