Genomic Imprinting Of Gtl2 In Somatic Cell Nuclear Transfer Cattle

Somatic cell nuclear transfer (SCNT) has great value in medicine, stock breeding and saving endangered animals, but the low efficiency of SCNT restricts it's application. The low efficiency of animal production using somatic cell nuclear transfer procedures is considered to be the result of an incomplete reprogramming of the donor somatic cell nucleus, which leads to a lack of, or abnormal expression of developmentally important genes. Most imprinted genes play important roles in embryogenesis and postnatal growth regulation. The development abnormalities of SCNT offspring are probably a result of abnormal expression of imprinted genes. However imprinting studies in SCNT cattle remain an underdeveloped area of research, largely because of the lack of information on sequences and polymorphisms of putative imprinted genes in the bovine.Gtl2 is one of imprinted genes in human and mouse, which regulates the translation of target mRNA as noncoding RNAs and plays an important role in regulating embryogenesis. In order to discuss the molecular mechanisms of the organ development defects and neonatal death in cattle produced by SCNT, we identified the Gtl2 expression patterns in six organs including heart, liver, spleen, lung, kidney and brain of catties produced by natural reproduction and SCNT.Expression patterns of imprinted genes are studied based on the fact that imprinted genes are monoallelically expressed. To study if gene expression is mono- or biallelic, a single nucleotide polymorphism (SNP) must be present to distinguish which parental allele is transcribed, which are DNA point mutations (base-pair change or insertions/deletions). Single strand conformation polymorphism (SSCP) is a reproducible, rapid and quite simple method and is widely used for the detection of SNP. We identified single nucleotide polymorphisms in Gtl2 through PCR-SSCP.Firstly, three pairs of PCR primers of Gtl2 were designed to amplify DNA fragments between 100 bp～300 bp through blasting the nucleotide sequences of the sheep (AY017220, AY017221, AY017222) and the cattle (XM872590). Secondly, we analyzed single nucleotide polymorphisms in Gtl2 through PCR-SSCP and identified hybrids through sequencing. Fortunately, hybrids were found in catties produced by natural breeding and SCNT to identify the Gtl2 expression patterns in six organs by RT-PCR-SSCP and sequencing. Our analysis demonstrated that, in cattle produced by SCNT, Gtl2 was monoallelic expression in heart and liver as shown in all six examined organs of cattle produced by natural reproduction. However Gtl2 was biallelic expression in brain, spleen, lung and kidney in cattle produced by SCNT. The result suggests that the abnormal expression of Gtl2 may contribute to the organ development defects and neonatal death in cattle produced by SCNT.