Simple and rapid method for routine screening of bovine leukemia virus by loop-mediated isothermal amplification assay.

Various techniques for screening and detection of bovine leukemia virus (BLV) were compared to ascertain a rapid and simple technique for routine examination. The performance of real-time PCR, nested PCR and loop-mediated isothermal amplification (LAMP) assays was compared using DNA extracted from whole blood instead of white blood cells (WBCs) of 23 cattle. Real-time PCR, LAMP and nested PCR detected 18, 16 and 11 BLV-positive cattle, respectively. These results suggest that LAMP using DNA from whole blood could enable rapid examination, as isolation of WBCs and electrophoresis is time-consuming and could be useful as a simple and rapid method for routine screening of BLV.

Aberrant CpG methylation of the imprinting control region KvDMR1 detected in assisted reproductive technology-produced calves and pathogenesis of large offspring syndrome.

Although somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) have the potential to produce genetically superior livestock, considerable numbers of abnormally large animals, including sheep and cattle affected by "large offspring syndrome" (LOS), have been produced by these assisted reproductive technologies (ART). Interestingly, these phenotypes are reminiscent of Beckwith-Wiedemann syndrome (BWS) in humans, which is an imprinting disorder characterized by pre- and/or postnatal overgrowth. The imprinting control region KvDMR1, which regulates the coordinated expression of growth control genes such as Cdkn1c, is known to be aberrantly hypomethylated in BWS. Therefore, we hypothesized that aberrant imprinting in this region could contribute to LOS. In this study, we analyzed the DNA methylation status of the Kcnq1ot1/Cdkn1c and Igf2/H19 domains on bovine chromosome 29 and examined the coordinated expression of imprinted genes surrounding them in seven calves derived by NT (which showed signs of developmental abnormality), two calves conceived by IVF (both developmentally abnormal), and three conventional calves that died of unrelated causes. Abnormal hypomethylation status at an imprinting control region of Kcnq1ot1/Cdkn1c domain was observed in two of seven NT-derived calves and one of two IVF-derived calves in almost all organs. Moreover, increased expression of Kcnq1ot1 and diminished expression of Cdkn1c were observed by RT-PCR analysis. This study is the first to describe the abnormal hypomethylation of the KvDMR1 domain and subsequent changes in the gene expression of Kcnq1ot1 and Cdkn1c in a subset of calves produced by ART. Our findings provide strong evidence for a role of altered imprinting control in the development of LOS in bovines.