ABSTRACT
The total RNA was extracted from porcine ovary. Porcine Follistatin cDNA was cloned by RT-PCR. Complete porcine follistatin cDNA coding sequences are presented including 1038 bp of open reading frame. The purified porcine follistatin cDNA was inserted into pGEX-4T-3 vector to construct the prokaryotic fusion protein expression vector. The recombinant expression plasmid was transformed into BL21 (DE3) and expression was induced by IPTG. Protein products were detected by SDS-PAGE and confirmed by Western blotting analysis, which showed that the yield of the Follistatin cDNA was a 63kD protein expression vector. Follistatin protein was expressed in the form of glutathione-S-transferase (GST) fusion protein in E. coli.
Subject(s)
Animals , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli , Genetics , Follistatin , Chemistry , Genetics , Molecular Sequence Data , Phylogeny , Recombinant Fusion Proteins , SwineABSTRACT
Despite recent successes in cloning various mammals and amphibians, the low efficiency of animals production and abnormal symptoms in many cloned animals are crucial problems in cloning technology. To overcome these problems, scientists focus on mechanisms of cloning. A possible cause of the low success frequency of cloning is the insufficient dedifferentiation and the inadequate reprogramming of the high differentiated adult somatic nucleus in enucleated oocytes, which caused by incomplete methylation and premature de novo remethylation of donor DNA. In cloned embryos the methylation level is higher than normal embryos, and this may cause aberrant expression of several important genes, especially imprinting genes. Study on these mechanisms is very important to improve the rate of successful cloned animals.
Subject(s)
Animals , Humans , Cloning, Organism , DNA Methylation , Genetics , Physiology , Epigenesis, Genetic , Genetics , Physiology , Genomic Imprinting , Genetics , PhysiologyABSTRACT
By the method of single preimplantation embryos differential display polymerase chain reaction (SPEDDRT-PCR), 25 reprogramming cDNA fragments were obtained from single 2-cell, 8-cell embryos and blastula. After cloning and sequencing, five of them were identified by reverse-Northern and characterized with stage-specific expression during reconstructed embryo development. This results will help to isolate full length reprogramming genes and study their function during embryonic development.