Study On Epigenetic Modification And Gene Expression Characteristics In Somatic Cell Cloned Goats

Although SCNT is a powerful tool used in mammalian cloning, its low efficiency has been observed in many species. Incomplete epigenetic reprogramming is suspected to cause the abnormalities and the low efficiency associated with SCNT. It is helpful to know epigenetic reprogramming threshold for proper development to understanding molecular mechanisms underlying SCNT animals surviving to adulthood. In present study, Southern hybridization, Real-time quantitative PCR and SSCP were used to investigate the methylation pattern of imprinting gene and repeated DNA, telomere length and the expression patterns of growth-related imprinting genes and non-imprinting genes in adult SCNT goats.1. Cloning and sequencing of DMRs of H19/IGF2 and IGF2R in goatIn present study, several pairs of primers were designed according to sequence of AJ566210 in sheep to amplify the imprinting domain upstream of H19 gene in goat. Following sequencing and overlapping, a 4.2kb sequence of H19/IGF2 DMR in goat was obtained(EF577239). The sequence upstream of hircine H19 was found to have the same structure as in sheep, mouse and human. These sequences fulfil the criteria of 4 CpG islands(with GC contents of 63％, 65％, 61％and 71％, respectively) and comprise multiple putative binding sites for the zinc-finger protein CTCE The four putative CTCF binding sites identified in the upstream region(sitesⅠ-Ⅳ) are all within conserved stretches of, 20-bp, and are positioned at 4079bp(siteⅠ), 3646bp (siteⅡ), 2866bp (siteⅢ), and 1221bp (siteⅣ) upstream of the transcription initiation site, respectively. Overall, the upstream sequences displays 93.5％identity with the syntenic sheep sequences.To obtain DMR in 2nd intron of goat IGF2R, primers were designed from reference sequence(AY182033) in sheep. Finally, a 2.8kb fragment was obtained and sequenced. Only 809bp of 5' end and 576bp of 3'end were obtained because of high GC content in the mid-region(EF577240). After confirmed by the BLAST engine, the obtained sequences share 95.7％identity with that of sheep. GC contents of the two sequences are 76％and 67％, respectively.2. Methylation status of H19 and IGF2R DMR in adult SCNT goatsThe methylation status of the putative CTCF sites, and of CpG dinuclcotides elsewhere in the upstream region of H19 in goat was investigated by digestion of 10μg genomic DNA with methylation-sensitive restriction enzymes, followed by Southern hybridization. The result revealed that the methylation level is not different in the cloned goats compared to the control animals(P＞0.05). Only one of cloned goats was out of the methylated range of controls. The methylation levels in adult clones derived from skin fibroblast was similar to that derived from ovary granulosa, and the average methylation status in fibroblast clones was lower than that in their nucleus donor.In order to investigate the methylation status at the intronic DMR of IGF2R, ear skin DNAs were digested with HindⅢand SacⅡhybridized with the IGF2R probe subsequently. The result suggested that, on average, the cloned animals had a significantly higher methylation ratio than the control animals(P＜0.05). The methylation levels in adult clones derived from skin fibroblast was similar to ovary granulose cells, and there was no difference between fibroblast clones and their nucleus donor.3. Methylation status of repeated DNA in adult SCNT goatsAfter digested with methylation-insensitive restriction enzyme MspI and methylation sensitive restriction enzyme HpaⅡ, the bulk DNA methylation status in adult SCNT clones was analysed using the minor satellite DNA probe HMP33.6. The result showed that the methylation level is not different in the cloned goats compared to the control animals (P＞0.05). The average methylation status in fibroblast clones was lower than that in granulose clones, and also lower than that in their nucleus donor. The methylation levels in granulose clones varied in a wide range(P＜0.01).4. Telomere length in adult SCNT goatsMean telomere length was determined by telomere restriction fragment(TRF) analysis. Isolated DNA(2.5-10μg) was digested using a HinfⅠ/RsaⅠenzyme mixture and then hybridized with a DIG-labeled Telomere Probe subsequently. The result suggested that mean terminal restriction fragment(TRF) lengths were not significantly different between the adult clones and age-matched controls(P＞0.05), but were varied in a large range in cloned goats. Goat clones reconstructed from skin fibroblasts displayed diverse telomere lengths, both shorter and similar to that from ovary granulose cells. The 2nd generation of clones also displayed diverse telomere lengths, both shorter and similar to controls (P＞0.05).5. Expression of growth-related imprinting gene in adult SCNT goatsIn present study, expressions of growth-related imprinting genes(H19, IGF2, and IGF2R) in adult SCNT goats were investigated by Real-time PCR. The expressions of these genes in adult clones were found largely normal but IGF2R was more highly expressed in cloned goats than in natural produced goats(P＜0.01). Analysis on mono-allelic expression pattern of imprinting genes indicated that mono-allelic expression patterns of H19 and IGF2 in cloned goats were similar to that in non-NT goats. The entire CDS sequence and the 3'UTR sequence of the IGF2 gene in goat was also obtained in present study. Analysis indicated that the open reading frame(ORF) of goat IGF2 is a 540 bp fragment encoding a 179-amino-acid protein. Compared with sheep and cattle's IGF2 cDNA, the identity from start codon ATG through poly(A) region is 99.47％and 97.68％, respectively. Compared with those of other ruminant species, putative amino acid sequence of IGF2 in goat is more homologous to that in sheep(98.88％identity) than in cattle(96.09％identity). The obtained GenBank accession numbers of H19, IGF2 and IGF2R are DQ666955, DQ645739 and DQ666954, respectively.6. Expression of growth-related non-imprinting gene in adult SCNT goatsThe expressions of growth-related non-imprinting gene IGF1, IGF1R, GHR and GHSR in adult SCNT goats were also confirmed by Real-time PCR. The results showed that IGF1R was more highly expressed in cloned goats than in controls(P＜0.01), while the expressions of IGF1, GHR and GHSR were fairly normal(P＞0.05). The obtained partial sequence of goat IGF1R gene was submitted to GenBank(DQ666953).