271 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A 35-DAY-OLD CLONED PIG FETUS USING THE ANNEALING CONTROL PRIMER SYSTEM

Abstract

Somatic cell nuclear transfer in pig has limitations due to the high incidence of fetal failure after embryo transfer to recipients. Reasons for the inefficient cloning are assumed to be due to abnormal and poorly developed placenta. Thus, this study was designed to determine possible genetic causes of neonatal deaths and other related abnormalities. Genes expressed specifically or prominently on Day 35 were identified in cloned pig placenta utilizing PCR technology regulated by annealing control primers (ACPs). The RNA was isolated using Trizol reagent. By utilizing 120 ACPs, 53 expressed sequence tags (ESTs) of genes that are differentially expressed in cloned pig placenta compared with normal placenta were cloned and sequenced. The cloned genes or ESTs exhibited significant sequence similarities to known genes or ESTs of other species. Ten of the total known genes, i.e., pregnancy associated glycoprotein, H19, 60S ribosomal protein L12, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, heme oxygenase 2, granulin precursor, and placenta-expressed transcript protein, were selected and their specific expression levels were confirmed by real-time RT-PCR in the normal and cloned pig placentas in triplicate using beta-actin for determining relative expression. The 60S ribosomal protein L12 and heme oxygenase 2 were highly expressed in the cloned pig placenta, whereas pregnancy associated glycoprotein, H19, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, granulin precursor, and placenta-expressed transcript protein were low or void. Our data suggest that the ACP system effectively identified tissue-specific genes in cloned pig placenta. Furthermore, identified genes would assist in developing insight into the genetic basis of fetal failure and help in resolving low pregnancy rate in the production of cloned pigs.