TTF-1 Gene Expression And Methylation In Lung Of Somatic Cell Nuclear Transfer Cattle

A variety of animals have been cloned successfully since 1997. But the low efficiency and the developmental defect, such as abnormal lung development, are the disadvantage of SCNT (somatic cell nuclear transfer). TTF-1 (Thyroid transcription factor-1) is a homeodomain transcription factor, a member of the NKX2 gene family, which is specifically expressed in thyroid, lung, and forebrain. It regulates the tissue-specific gene expressions of thyroglobulin, surfactant proteins et al. TTF-1 plays a pivotal role during lung development and controls the homeostasis of lung in adult. It's helpful to elucidate the mechanism of lung disfunction of SCNT animals to investigate the mRNA expression level and methylation profile of bovine TTF-1.Bovine TTF-1 was cloned by means of RT-PCR, inverse PCR, and degenerate PCR; the total RNA of 4 cloned cattles (9C1, 9C3, 9C4, and 9C8) and 2 controls (9N1 and 9N4) were extracted frome lung, and the expression of TTF-1 gene is investigated with quantitative real-time RT-PCR; and the methylation profile of bovine TTF-1 gene sequence upstream of the translation initiator was measured. Results were as following:1. Bovine TTF-1, including exon1 upstream sequence 135bp, exon1 77bp, intron1 709bp, exon2 373bp, intron2 932bp and partial exon3 273bp, was cloned and sequenced.2. The expression of bovine TTF-1 gene in lung tissue was determined with quantitative real-time RT-PCR. The mRNA levels in SCNT cattles of 9C1, 9C3, 9C4, 9C8, and in controls of 9N1 and 9N4 were 7.95, 0.81, 2.94, 1.53, and 6.66, 4.43, respectively.3. The methylation profile of three CpG islands containing 50 CpG dinucleotides was investigated. Compared to control (9N1), SCNT cattle (9C3) with lung defect did not show higher level of methylation. Most of the CpG dinucleotides are unmethylated.4. The 5'upstream sequence of bovine TTF-1 gene in cloned cattles 9C3, 9C8 and in controls 9N1, 9N4 was cloned and sequenced respectively. The result of sequence alignment shows that, the nucleotide -881 in cloned cattle 9C3, 9C8, and in control 9N1 was A, and in control 9N4 was G; the nucleotide -741 in cloned cattle 9C3, 9C8, and in control 9N1 was C, and in control 9N4 was A; the nucleotide -595 in cloned cattle 9C3, 9C8, and in control 9N1 was C, and in control 9N4 was C; the nucleotide -551 in cloned cattle 9C3, 9C8, and in control 9N4 was C, and in control 9N1 was T; the nucleotide -471 in cloned cattle 9C3, 9C8, and in control 9N1 was A, and in control was T; the nucleotide -311 in cloned cattle 9C3, 9C8, and in control 9N1 was G, and in control 9N4 was A; the nucleotide -281/-299 in cloned cattle 9C3, 9C8, and in control 9N1 was T, and in control 9N4 was C; the nucleotide -97 in cloned cattle 9C8, in control 9N1 and 9N4 was C, and in 9C3 was T. And all of the nucleotide substitutions were located out of the transcription factor binding site.