Histone Acetylation Study In Lung Of Somatic Cloned Cattle

The first mammal have been successfully cloned from a differentiated animal cell was the sheep "Dolly" in 1997, and other subsequent success with a range of mammal somatic cloned animal, such as mice, cattle, goat, pig, horse and mule etc. Despite this, the mammal somatic cloning is still inefficient. Clones are lost from the earliest developmental stages and throughout pregnancy. Now more and more studies support the evidence on the failure of cloning: chromatin inappropriate epigenetic reprogramming, and then result in abnormal genes expression. Epigenetic modification of the genome ensures proper gene activation during development and involves: genome methylation changes; the assembly of histon and histone variants into nucleosomes and remodeling of other chromatin-associated proteins such as linker histones, polycomb group, nuclear scaffold protein, and transcription factors. Histon acetylation plays an important role in regulation chromatin structure and transcriptional activity. However, there is a little investigation in cloned cattle about histone acetylation. Andmore, in many dead cloned cattle, the lung abnormality is mostly obvious. So, in our studies, we want to know how about the histone acetylation in the lung of cloned cattle.In the histone acetylation study, we used the histone immunoprecipitation assay and real-time quantity PCR method. We analyzed 18 samples (including 9N1, 9N3, 9N4, 8C1, 8C2, 8C3, 8C4, 9C1, 9C2,9C3,9C4, 9C5, 9C6, 9C8,9C12, Jiumei, Benben and Xiaobai) with 15 genes (including b-Actin, BMP4, EGF, FGF, FGFBP, FGFR, GH, GHR, GIi2, HGF, HGFR, 1GF1, RAR, SHH and VEGF). We get to the conclusions: (1) the histon acetylation level has some derease in the lung of cloned cattle; (2) in the cloned cattle Xiaobai, the histone acetylation level is lower than that in normal control, however, higher or adjacent to that in other dead cloned cattle; (3) there is no significant difference during those cattle from the cumulus, fetal fibroblast and adult fibroblast cell donor.We also analyzed some genes' expression, including 13 samples (9N1, 9N3, 9N4, 8C1, 9C3, 9C4, 9C5, 9C6, 9C8, 9C12, Jiumei, Benben and Xiaobai et al) and 7 genes (BMP4, FGF10, FGFBP, FGFR, 1GF1, SPA and VEGF et al). We find some genes' expression level increase, such as BMP4, IGF1, SPA and VEGF, and others decrease, such as FGF10, FGFBP and FGFR.However, it is difficult to relate the histone acetylation level with the gene expression level. We indicate, although histone acetylation acts to enhance the access of transcription-associated protein to DNA, the activation of gene expression only need containing some acetylation level, but the hyperacetylation and underacetylation would effect the gene expression, so as to the abnormal development.In order to investigate the histone acetylation in gene different regions, we used normal cattle 4N1, 6N1, 9N3, cloned cattle Xiaobai and 9C4, with the IGF1 and GH genes. We designed 14 and 8 pairs of primers on their DNA sequences, respectively. We suggest: (1) histone must maintain some acetylation level whether the associating-gene is activated or inactivated; (2) acetylation and deacetylation are global in these genes' chromosomal domains; (3) but uneven; (4) and the level will change according to the development stage.