Preliminary Studies On Buffalo Mammary Gene Expression Profile And Growth Hormone Transgenic Buffalo

The swamp buffalo, roughage resistance and good quality milk, is a great potential livestock for development in south of China. However, due to the poor milk yield performance of local buffalo, China’s buffalo industry is in a slow development. Therefore, how to use modern bio-technology to breed higher milk yield buffalo is the primary and major goal. In this study, gene expression profile construction of the buffalo lactation and non-lactating mammary gland was used to analyze differential expression genes, which could be useful in buffalo lactation molecular mechanisms and looking for the special promoters. These promoters could regulate exogenous gene expression in the mammary gland with the goal of higher milk yield buffalo. Then, transposon system was explored in buffalo’s transgenic reseach with a high transgenic efficency. The vectors expressing extraneous growth hormone gene (GH) were transfected buffalo mammary epithelial cells and mammary tissue to dectection milk protein gene expression. At the last, random integration and transposable active integration transgenic methods were used to carry recombinant growth hormone gene (GH) regulated by lactation mammary-specific expression promoter. Higher milk yield buffalo is the final target. The main results of this study are summarized as follows:1. Differential gene expression between buffalo lactation and non-lactation mammary gland. Gene expression profile construction of the buffalo lactation and non-lactating mammary gland was used to analyze differential expression genes, which could be useful in buffalo lactation molecular mechanisms and looking for the special promoters. These promoters could regulate exogenous gene expression in the mammary gland with the goal of higher milk yield buffalo. The high-quality reads obtained by RNA-seq were assembled and mapped to114,014single Unigenes, which were mapped to270pathways from KEGG database respectively. The genes associated with milk lipid, milk protein and milk lactose synthesis and metabolism pathways expressed in a high amount. Genes expressed only in the lactation mammary gland were3,053accounting for2.68%of all genes, which were37,162genes accounting for32.61%in non-lactation mammary gland. The expression up-regulated genes in lactation mammary gland had1390and down-regulated expression genes were5851. There were228of270pathways which have differential expressing genes, such as JAK/STAT and MAPK singnal pathway. Quantitative real-time PCR (QRT-PCR) was used to confirm the results of RNA-seq. Kappa casein (CN3) was the most expression at these two periods. The expression of CN1S1gene increased6958times in lactation period compared to non-lactating period. The gene expression of kCN and aLA increased about1000times,4800times for CN1S2and244times for beta casein respectively. There were expression of GHR, PRLR and IGF1R detected in two period’s mammary gland, which may indicate that GH、PRL and IGF1play roles in molecular regulation of milk secretion.2. Gene expression of buffalo immortalized mammary epithelial cells (BME). BME cells immortalized by pXL-BACII-bCN2/LT vector, which large T antigen gene regulated by buffalo’s beta casein gene promoter in apiggyBac transposon vector, reduced the loss of acinar-like cells and fibroblast-change process. With prolactin induced, milk protein gene expression could be detected in the30th generation immortalized BME cells. Comparison to gene expression of non-lactation mammary tissue, the increase gene expression times of CN1S1gene were88.32, which were32.09times for CN3gene and24.99times for a LA gene. These results indicate that the cell detection model of transgenic mammary gland expression vector was established preliminarily.3. Establishment of transposon transgene system in buffalo. Transposon transgene vector from piggyBac (PB), passport (PP) and sleeping beauty (SB) were constructed and transposon system was explored in buffalo’s fetal fibroblast for transgenic reseach with a high transgenic efficency. The maker gene expression of EGFP in buffalo fetal fibroblast (BFF) could be observed after transfected by transposon vector. The G418-resistent cell colony formation numbers (CFN) were not different among p18T vector、PB、PP and SB transposon, which carried the Neo maker gene. Comparison with CFN of random integration transgenic methods, CFN obtained of PB transposon system was22times, PP transposon system was8times and SB transposon system was14times. PB transposon system had the most transgenic efficiency. The transgene integration copy number was detected by QRT-PCR. The copy number of random integration group varied from5to51.9copies per haploid genome, which were16.62copies for PB transposon and2.5copies for PP transposon. SB transposon with98copies was the most number of transgene integration copies. These results indicate that three transposon transgene systems could applied in buffalo’s transgenic research, which transgenic efficency is better than random integration transgene.4. Construction and detection of growth hormone transposon vector. Full-length genomic DNA fragments of human GH (2180bp), cattle GH (2207bp) and buffalo GH (2192bp, Gi:JF894306) were amplified from blood genome DNA. GH gene and amino acid were conservative in mammary animal. Human breast cancer cells (Bcap37) were transfected pXL-BacII-bCN2.8/bGH2.1-EGFP-Neo or PXL-BacII-bCN2.8/hGH2.2-EGFP-Neo vector, which carried human GH gene or cattle GH gene, with transposase vector pCMV/PBase-bPGK/DsRed. Transgenic GH gene expressed in G418-resistent Bcap37cells confirmed by RT-PCR and western blot. Immortalized BME cells, which transfected p18T-bGH or PB-bGH transgenic vector, could be detected the bovine GH gene expression and the significant expression increase of beta casein gene, CN1S1casein gene and alpha-lactalbumin gene. The gene expression of GHR gene and IGF1R gene were up-regulation. Moreover, gene expression of CN1S1, CN1S2, beta casein and kappa casein increased more than50times in mammary tissue transfected with pB-bCN2.8/bGH2.1-EGFP-neo vector. These results showed that transfected transgenic vector could increase milk protein gene expression preliminary.5. Production of GH transgenic clone buffalo embryo and detection of premature birth transgenic buffalo. Transgenic embryos, produced by oocyte cytoplasm injection and inner cytoplasm sperm injection (ICSI) with pXL-BacII-bCN2.8/bGH2.1-EGFP-Neo and transposase vector, could be observed EGFP expression. BFF cells transfected vector and selected as positive cell colony by G418, which were the nucleus donor for somatic nuclear transfer. The nuclear transfer, p18T-bCN2.8/bGH2.2-hEF1a/EGFP-IRES-neo transgenic BFF cells as nuclus donor, reconstructed blastocyst could be observed EGFP expression. A premature buffalo offspring after embryo transplant was born at pregnant9months. The premature offspring could be observed green fluorescence at head side under the specific blue light. This offspring’s heart, lung, liver, stomach and spleen tissues were normal development. But large intestine, kidney and reproductive system were dysplasia, which were adhesions. Green fluorescence was observed in frozen sections of the heart, liver, spleen, lung, diaphragm and muscle tissue under laser scanning confocal microscope. Transgene were integrated in the premature offspring’s genome detected by PCR and chromosome walking. These results laid a good basis for breeding mammary-specific expression GH transgenic buffalo.