| Prolactin (PRL), a peptide hormone, could improve the development of mammary gland, promote and maintain the lactation. The physiologic function of PRL is mediated by the PRLR-JAK/Stat5 signal pathway, PRL, PRLR, Stat5a and Stat5b genes are the essential factors in this pathway, which mediate the mammary gland development and lactation. Stat5a and Stat5b are intracellular signal transductors, which form homo or hetero dimers to regulate the expression of target genes. It was reported that Stat5a was one of the main transductors involving in the regulation of the proliferation and differentiation of mammary gland under the influence of PRL gene, but its detailed regulation mechanism was unknown. To investigate if it is feasible to improve the milk yield or milk quality of buffalo by expressing buffalo PRL gene in the mammary gland, in present study, the transgenic vectors of expressing PRL gene specially in the mammary gland were constructed and screened, mouse model of mammary specially expressing PRL gene was generated and analyzed. In addition, the regulation mechanism of transcription factor Stat5a on mammary cell proliferation and differentiation was investigated by searching its target genes.1. Construction of mammary gland-specific PRL expression vectorIn order to select vectors suitable for nuclear transfer and pronuclear injection methods respectively, using the PRL-special primers with a His tag and a thrombin recognition site added, a PRL fragment of 804 bp length without signal peptide was obtained using total RNA from adult female pituitary tissue of buffalo as RT-PCR template. Using pMD18-T vector as the backbone, which contented BCNpolyA fragment between KpnI and SacI sites, five mammary gland-specific PRL expression vectors were constructed through inserting the PRL fragment, beta-casein (BCN) promoter fragment(3.8BCN or 5.2BCN) and the marker gene or screen gene (cmv-EGFP-SV40polyA-SV40polyA, cmv-EGFP-SV40polyA-SV40-neo-SV40polyA or cmv-EGFP-IRES-neo-SV40 polyA). Five vectors were transiently transfected into the Bcap-37 cells to analyze the expression of foreign genes respectively. Except the p3.8BCN-PRL-BCNpolyA-cmv-EGFP-SV40polyA-SV40-neo vector, other four vectors could express foreign PRL gene in transfected Bcap-37 cells. The results of western blot analysis showed the vectors containing the 5.2BCN promoter could express exogenous PRL prolactin higher than that of the 3.8BCN promoter in the transfected cells. Considering the demands of the different transgenic animal operation methods, the p5.2BCN-PRL-BCNpolyA-cmv-EGFP-SV40polyA vector was selected to produce transgenic animals through cytoplasmic injection method, the p5.2BCN-PRL-BCNpolyA-cmv-EGFP-IRES-neo-SV40polyA vector was more suitable to screen the transgenic positive cells during the nuclear transfer process.2. Establishment and analysis of the PRL transgenic mouse modelThe linearized p5.2BCN-PRL-BCNpolyA-cmv-EGFP-SV40polyA vector was microinjected into the pronucleus of fertilized FVB mouse eggs, and 8 of transgenic FO mice were obtained. Clear green fluorescence signal was observed in some of FO transgenic mice by both of in vivo fluorescence and claw tissue fluorescence detection. The PCR results of PRL and cmv fragments showed that a 752 bp PRL fragment and a 599 bp cmv fragment were amplified in some of F1 mice. The results of Southern blot analysis further confirmed that the positive mice detected by genome DNA PCR analysis indeed had the integration of foreign gene. The green fluorescence signal was observed in some of F2 transgenic mice under the long-wave UV lamp. The results of the copy number detected by Q-PCR analysis showed that more than ten copies of exogenous gene were integrated into the genome of transgenic mice, which had clear green fluorescence signal under the long-wave UV lamp, while only 2 copies were integrated into the genome of transgenic mice, which had no clear green fluorescence signal. The results of the foreign gene integration site analysis showed that there was no other gene in the range from upstream 20 kb to downstream 20 kb of the foreign gene integration site in the mouse which had clear green fluorescence signal by gene walking method. The above results indicated that it was feasible to produce transgenic PRL mice by pronuclear injection method, and the exogenous gene could steadily inherit in the transgenic mice offspring.The expression level of the foreign PRL protein in the transgenic mouse and the influence of PRL expression on the reproduction and the safety of transgenic mouse were further analyzed. The results of Western Blot analysis of the main tissues of transgenic mice showed that the exogenous PRL protein only expressed in the mammary gland and milk. The content of PRL protein in milk of the transgenic mice detected by ELISA analysis was 611.74～2773.96 μIU/ml, 77.92～706.80% times higher than that of in the wild-type mice milk. The average content was 769.24 μIU/ml,223.73% times higher than that of in the wild-type mice. The results of QRT-PCR analysis showed the relative expression of milk protein (CNS2) and lactose (B4galtl) increased significantly (P≤0.01) in milk of transgenic mice. The PRL protein content in the serum of transgenic mice was significantly higher than that of wild-type mice during the lactation period by ELISA analysis (P≤0.01), while during the non-lactation period, there was no significant different between transgenic and wild-type mice (P≥0.05). The above results reveal that the foreign PRL gene could express specifically in the mammary gland of transgenic mice, its expression would improve the relative expression of milk protein-and lactose-related genes, and has no side-effect on the survival and reproductive ability of transgenic mice.3. Screening and analysis of Stat5a target genesTo find out the target genes of the transcription factor Stat5a, based on the results of the expressing level of Stats genes in the different developmental states of mammary gland by QRT-PCR analysis, G-NC, G-PRL and L-PRL samples were analyzed by ChIP-seq and RNA-seq respectively, and formed G-NC VS G-PRL and G-PRL VS L-PRL,2 comparison group data. In the data of ChIP-seq analysis, over 25M total Reads, including uniquely mapped Reads over 20M were obtained from each of the 3 samples, which was more than 78% of total Reads.218,234 and 160 Peaks were obtained respectively for 3 samples, the average length of peaks was about 1kb. The results of Gene Ontology analysis of the Peaks showed these genes were most relative with cellular process, metabolic process and single-organism process in the term of biological process, also very close with binding, catalytic activity and molecular transducer activity in the term of molecular function. Over 11M total Reads including uniquely mapped Reads over 8M were obtained in RNA-seq data of each sample. The results of RNA-seq data analysis showed that the number of detected genes tended to saturation, the distribution of Reads covered almost all reference genome and the Reads distributed randomly on the genes, which indicated that high-quality data of RNA-seq was obtained. The results of differential expression genes analysis showed that when comparing the G-PRL with G-NC sample,253 genes had up-regulated expression, and 414 genes were down-regulated expression, while comparing the L-PRL with G-PRL sample, 157 genes had increased expression, and 2180 genes had decreased expression. The results of Gene Ontology analysis showed the differential expressed genes in 2 groups were most relative with cellular process, metabolic process and biological regulation in the term of biological process, and also closed with binding, catalytic activity and molecular transducer activity in the term of molecular function. The GO analysis results were similar with that of the Peaks analysis in ChIP-seq data, which demonstrated that the binding of Stat5a was closely related with the gene expression level neighbored with the Stat5a binding sites.Total 10 target genes regulated by Stat5a were found by combining the data of ChIP-seq and RNA-seq. When comparing G-PRL with L-PRL sample, it was found that Stat5a bound respectively with its binding site in the Erbb4, Tnfrsf13c, Gabrp, Arrdc4, Pvtl and AI848285 genes to up-regulate these genes’ expression in the later pregnant period. Stat5a bound respectively with its binding site 1 in the Lgr6 and Ptprv genes to up-regulate the expression of the two genes in the late pregnant period, while bound respectively with its binding site 2 in these genes to down-regulate the gene expression in the lactation period. When comparing G-NC with G-PRL sample, it was found that in the late pregnant period, Stat5a bound respectively with its binding site 1 in the Ceacam2 and Atp2b2 genes to down-regulate the expression of these genes in the wild-type mice, while due to the expression of the foreign PRL gene, Stat5a could not bind respectively with its binding site 1 in the Ceacam2 and Atp2b2 genes to up-regulate the expression of these genes in the transgenic mice. These results confirmed that, by binding with its binding site, Stat5a up-regulated the expression of Erbb4, Tnfrsfl3c, Lgr6 genes etc. in the late pregnant period, which related with the function of mitosis, apoptosis and differentiation etc., and participated in the regulation of proliferation and differentiation of breast cells through the signal pathway of these target genes, including Shc/Grb2/Soc-Rac-Raf-MPK, NF-KAPPA and Wnt etc.The above results indicate that it is feasible to improve the milk yield or milk quality of transgenic animal by expressing PRL gene specifically in the mammary gland. Stat5a, a PRL intracellular signal factor, participates in the regulation of proliferation and differentiation of breast cells through regulating the expression of target genes, for example Erbb4, Tnfrsfl3c, Lgr6 etc. |
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