Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Expression of Human GCSF in Mammary Gland of Mice by Injection of Plasmid DNA

The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency in transgenic animals. The expression vector of mammary gland (pINGG) containing GCSF genomic DNA was injected into mouse mammary gland, and expression was detected in the milk of mice. The result showed that mammary gland injection method could provide a convenient transient system to confirm vector validity.

THE EXPRESSION OF APOPTOSIS RELATED GENES IN THE PROCESS OF CANCERATION OF ATYPICAL HYPERPLASIA OF MAMMARY DUCT

Objective： To investigate the expression of apoptosis related genes p53 and bcl-2 in atypical hyperplasia of mammary duct and the relationship between the gene expression and oncogenesis of breast. Methods： mRNA of apoptosis related gene p53 and bcl-2 were detected by in situ hybridization in 44 cases of atypical ductal hyperplasia. p53 protein expression was detected by immunohistochemistry. The data were compared with those of 6 cases of benign hyperplasia and 26 cases of breast carcinoma. Results： The expression of p53 mRNA was 66.7% in benign hyperplasia, 40% in atypical ductal hyperplasia （55.6% in mild, 41.7% in medium, 26.1% in severe） and 19.2% in carcinoma （of which 21.4% were intraductal carcinoma and 16.7% were invasive）. The expression of p53 protein was negative in benign hyperplasia, 24% in atypical hyperplasia （mild 11.1%, medium 25%, severe 34.8%）, 38.5% in carcinoma （intraductal carcinoma 35.7%, invasive ductal carcinoma 41.7%）. The expression of bcl-2 was negative in benign hyperplasia, 78.6% in intraductal carcinoma, 83.3% in invasive ductal carcinoma. Conclusion： Loss and mutation of p53 gene and excessive expression bcl-2 mRNA were detected in severe atypical ductal hyperplasia.

Stable Expression of Antibacterial Peptide CecropinB in Dairy Goat Mammary Gland Epithelial Cells

The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.

Effect of exogenous somatotropin and staged ovariectomy on mRNA expression of select ECM-related proteins in mammary tissue of prepubertal Holstein calves

Growth hormone (GH) and estrogen are essential stimulators of mammary cell proliferation and mammary development as mammals near puberty. Mammary ductal growth requires modifications of the extracellular matrix (ECM) for this tissue expansion to occur. Our purpose was to evaluate the effects of exogenous GH and ovariectomy (known to impact estrogen production) on gene expression of selected ECM proteins in the mammary parenchyma (PAR) and mammary fat pad (MFP) of prepubertal calves. Our hypothesis was that both GH and ovariectomy would alter the mRNA expression of multiple mammary ECM proteins. However, treatment with GH significantly reduced the expression of only fibronectin in PAR. However, the mRNA expression of all of the ECM proteins tested was numerically lower in PAR from GH treated calves. In contrast, staged ovariectomy decreased expression of fibronectin and heat shock protein 90 but increased expression of epimorphin in mammary PAR. In the MFP expression of Rac-1 and fascin were increased. These findings suggest that effects of exogenous GH on mammary gland composition are only marginally dependent on alterations in ECM proteins but the more pronounced effects of ovariectomy (reduced PAR mass and altered myoepithelial ontogeny) are more likely linked to changes in expression of ECM proteins.

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.

Identification of Differentially Expressed MicroRNAs During the Development of Chinese Murine Mammary Gland

MicroRNAs （miRNAs） are endogenous -22 nucleofide-long noncoding RNAs. In this study, to investigate miRNA expression profiles and their functions in mammary gland development, we have used microarray as well as qRT-PCR, to analyze the miRNA expression changes along the murine mammary cycle during pregnancy, particularly on transition from pregnancy to lactation. It shows that every developmental stage of the mammary gland has its own mjRNA expression pattern. Compared with virgin and involution, some miRNAs such as miR-138 and miR-431 are downregulated, whereas, some miRNAs such as miR-133 and miR-133a-133b are upregulated during pregnancy and lactation. These results indicate that miRNAs are functionally involved in mammary gland development.

Bzw2 Promotes Proliferation and Lactation of Mammary Epithelial Cell in Dairy Goat

Mitosis of mammary epithelial cell is foundation of mammal lactation. We developed a strategy of combined application of generation of longer cDNA fragments from the serial analysis of gene expression （SAGE） tags for gene identification （GLGI） to screen and identify genes influencing lactating ability of mammary epithelial cell in dairy goat. GLGI as a new tag identification technique was brought about with SAGE. Bzw2 was found as a candidate gene related to lactation by screening Long-SAGE library of mammary gland in dairy goat. Bzw2 cDNA was synthesized by switching mechanism at 5＂-end of RNA transcript （SMART） technology. The mRNA level of Bzw2 was relatively higher in early lactation than in other development stages of mammary gland. The proliferation of mammary epithelial cell was inhibited by transfecting specific shRNA of Bzw2. The mRNA levels of Stat5, Csn2 and Prlr were also down-regulated, suggesting the lactating ability of mammary epithelial cell was attenuated after Bzw2 RNAi. The reduction of mammary epithelial cell growth and lactation by Bzw2 RNAi was rescued through over-expression of Bzw2. These results revealed that Bzw2 might play an important role in lactation though the molecular mechanism was still unclear.

柯乐猪异常乳腺组织的TMT蛋白组学分析

【目的】基于串联体量标记(TMT)定量蛋白组学技术探讨柯乐猪母猪乳腺发育的调控机制,为选育工作提供依据。【方法】选取乳腺发育正常和异常的柯乐猪母猪各3头,采集乳腺组织,利用TMT定量蛋白组学技术探究正常和异常乳腺组织的差异表达蛋白,筛选与乳腺发育相关的差异关键蛋白,并对差异表达蛋白进行生物信息学分析。【结果】从柯乐猪乳腺组织中共筛选出474个差异表达蛋白,包括245个上调蛋白、229个下调蛋白;基因本体(GO)功能注释和富集分析显示,差异表达蛋白主要富集在上皮细胞分化、皮肤形成和肾小管上皮细胞分化等生物学过程中,主要分布在胞外区和细胞外空隙中,参与调节肽酶抑制剂活性和肽链内切酶活性;京都基因与基因组百科全书(KEGG)富集分析显示,差异表达蛋白主要富集在补体系统、过氧化物酶体增殖物激活受体和金黄色葡萄球菌感染等信号通路上;结合差异表达蛋白互作网络分析,筛选出10个核心差异表达蛋白[上调蛋白有P14287(骨桥蛋白)、F1RXF9(角蛋白25)、I3LDS3(角蛋白10)、Q75QW1(上皮细胞黏附分子)、A0A5S8KLN1(丛生蛋白)、F1RW75(桥粒素),下调蛋白有A0A286ZT13(白蛋白)、P06867(血纤维蛋白溶酶原)、F1SCC9(含丝氨酸蛋白酶抑制剂结构域的蛋白)、P50447(α-1-抗胰蛋白酶)]。【结论】采用TMT定量蛋白组学技术能有效筛选出母猪正常和异常乳腺组织中的差异表达蛋白。

采食高淀粉高油日粮奶牛的泌乳性能、乳成分变化及乳腺差异表达基因筛选

【目的】研究高淀粉高油日粮对奶牛泌乳性能和乳腺组织转录组的影响,探索乳脂下降综合征(Milk fat depression,MFD)的分子机制。【方法】选用8头荷斯坦泌乳奶牛,随机分为对照组和处理组(4头/组),采用2×2反转试验设计,分为2期,每期23 d,期间2组对调。每期的前16 d,对照组奶牛饲喂低淀粉低油的基础饲粮,对照组日粮的泌乳净能为6.78 MJ/kg;处理组奶牛在基础饲粮基础上添加266 g/kg(DM基础)细粉碎玉米和46 g/kg(DM基础)大豆油;后7 d 2组奶牛均喂基础饲粮,处理组日粮的泌乳净能为7.66 MJ/kg。分别于每期的第1、4、7、10、13、16、19和22天测定产奶量和乳成分,第16天采集乳腺组织用于检测转录本的变化。【结果】与对照组相比,处理组第13、16天奶牛的干物质采食量和产奶量显著降低(P<0.05);饲喂高淀粉高油饲粮7 d后乳脂率开始降低,其中,第10、13、16和19天的乳脂率和乳脂产量显著降低(P<0.05);第13、16和19天的乳蛋白、乳糖和非脂固形物含量显著提高(P<0.05)。在奶牛乳腺对照组和处理组中共检测到235个显著差异表达的基因,其中,64个上调、171个下调。GO分析表明,差异表达基因主要与炎症反应、α−氨基酸代谢、有机氮化合物代谢、细胞发育、脂质生物合成和脂肪细胞分化等相关;KEGG分析表明,差异表达基因主要与轴突导向、NF-κB信号通路、钙信号通路等相关。结合差异表达基因与泌乳性能的相关性分析,本研究筛选出了部分与脂质调控相关的基因作为研究奶牛MFD状态下脂质代谢的候选基因,包括在乳腺中下调表达的FGFR4、VDR、HTR2B、CCL21和TYRP1。【结论】高淀粉高油饲粮饲喂奶牛降低了干物质采食量、产奶量和乳脂率,提高了乳蛋白、乳糖和非脂固形物含量,下调了奶牛乳腺中脂肪酸合成相关基因的表达。本研究为科学配制日粮和研究MFD的分子机制研究提供了数据参考。

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin,lactation and dry periods.A total of 122 genes were identified as differentially expressed,including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods.Gene ontology analysis showed the functional classification of the up-regulated genes in lactation,including transport,biosynthetic process,signal transduction,catalytic activity,immune system process,cell death,and positive regulation of the developmental process.Microarray data clarified molecular events in bovine mammary gland lactation.

脂多糖与钙离子共处理的牛乳腺上皮细胞miRNA表达谱的比较分析

【目的】探索钙离子对牛乳腺上皮细胞炎症反应的调控作用,比较分析脂多糖与钙离子共处理的牛乳腺上皮细胞的miRNA表达谱。【方法】使用脂多糖处理牛乳腺上皮细胞构建炎症反应模型,检测不同浓度(0.5~6.6 mmol/L)钙离子和脂多糖(50μg/mL)共处理的牛乳腺上皮细胞的炎症因子转录水平。提取正常、脂多糖处理、脂多糖与钙离子共处理牛乳腺上皮细胞样本中总RNA,每组各3个样本。对9个RNA样本进行质量评估,并对其开展miRNA测序和生物信息学分析,最后对3组共有差异表达miRNA进行实时荧光定量PCR验证。【结果】在牛乳腺上皮细胞炎症模型中,高浓度钙离子可抑制炎症因子的转录。9个样本的clean reads在raw reads中占比为93.86%~98.18%,序列分布长度均集中在21~24 nt之间。3组样本的组内相似性高,而组间差异度高。3组样本共鉴定出47个差异表达miRNA,共有差异表达miRNA分别为bta-miR-11980、bta-miR-1246和bta-miR-677。GO功能和KEGG通路富集分析显示,差异表达miRNA的靶基因显著富集于细胞通讯调节、信号调节、内膜系统、系统发育、细胞发育过程、细胞分化等GO条目,显著富集于HIF-1信号通路、ErbB信号通路、TNF信号通路、突触囊泡周期、肌醇磷酸代谢途径等通路。实时荧光定量PCR验证结果显示,3组样本共有差异表达miRNA(bta-miR-677和bta-miR-1246)表达趋势与转录组测序结果一致。【结论】钙离子通过调控牛乳腺上皮细胞内bta-miR-11980、bta-miR-1246和bta-miR-677等差异表达miRNA的表达模式,间接影响牛乳腺上皮细胞炎症反应进程。

Gene expression profiling in porcine mammary gland during lactation and identification of breed- and developmental-stage-specific genes

A total of 28941 ESTs were sequenced from five 5′-directed non-normalized cDNA libraries, which were assembled into 2212 contigs; 5642 singlets using CAP3. These sequences were annotated; clustered into 6857 unique genes, 2072 of which having no functional annotations were considered as novel genes. These genes were further classified into Gene Ontology categories. By comparing the expression profiles, we identified some breed-and developmental-stage-specific gene groups. These genes may be relative to reproductive performance or play important roles in milk synthesis, secretion; mammary involution. The unknown EST sequences; expression profiles at different developmental stages; breeds are very important resources for further research.

Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.

Expression and regulation of hFIX minigene and cDNA driven by β-casein gene in mouse mammary gland

Mammary gland specific expression vectors for human clotting factor IX (hFIX) and LacZ reporter gene driven by bovine β\|casein gene were constructed. Vectors were packaged by stearylamine (SA) liposome and were transferred to lactating mice via tail vein. Both hFIX and LacZ gene could be expressed in the mammary gland of the treated mice. The highest production of hFIX protein was 80.28 ng per mL milk, and more than 85% of hFIX protein appeared to be γ carboxylation and biologically active. The results suggested that the 2.0 kb sequence of β casein gene including promoter, exon 1 was effective to drive hFIX gene expression in mammary gland and intron 1 of β casein gene had an effect on the tissue specific expression. The expression level in mouse milk injected with hFIX minigene vector containing hFIX endogenous intron 1 was increased by above 3 times of that injected with hFIX cDNA vector.

荷斯坦奶牛DPP6和PRKN基因的生物信息学及表达规律分析

【目的】旨在探究前期GWAS筛选出的与荷斯坦奶牛产奶相关的二肽基肽酶样6(Dipeptidyl Peptidase⁃like 6,DPP6)和E3泛素连接酶基因(Parkin gene,PRKN)的生物学性质及表达特性,为后续验证DPP6和PRKN基因对于荷斯坦奶牛相关调控机制研究提供基础。【方法】利用ProtParam、Phyre2、NetPhos 3.1等软件分析DPP6和PRKN的理化性质与蛋白结构,并采用实时荧光定量PCR(Real⁃Time quantitative PCR,RT⁃qPCR)检测DPP6和PRKN基因在荷斯坦奶牛的心脏、脾脏、肝脏、肺脏、肾脏、乳腺、子宫、卵巢、瘤胃、黄体10个组织中的表达规律;通过脂多糖(Lipopolysaccharide,LPS)诱导乳腺上皮细胞(bMECs),初步检测DPP6和PRKN基因在炎症反应中的表达模式。【结果】DPP6蛋白编码863个氨基酸,与山羊的亲缘关系最近;PRKN蛋白编码488个氨基酸,其中甘氨酸含量最多,其序列与马鹿的相似性最高,保守型较强。本研究推测2个物种的DPP6和PRKN蛋白可能具有相似的生物学功能,并且2个蛋白均属于不稳定亲水性蛋白。DPP6和PRKN基因在组织中普遍表达。其中,DPP6基因在乳腺、肺脏、脾脏以及肾脏组织中的表达量显著高于其他组织,而PRKN基因在乳腺、肾脏、瘤胃组织中显著高表达。此外,相比于空白对照组,DPP6与PRKN基因均能够在LPS诱导的bMECs中极显著表达(DPP6基因极显著下调,PRKN基因极显著上调)。【结论】DPP6与PRKN基因可能在调控荷斯坦奶牛乳房炎症过程中发挥潜在作用,为后期荷斯坦奶牛DPP6与PRKN两个基因在奶牛bMECs炎症反应相关调控机制研究提供理论依据。

The effects of thyroid-stimulating hormone,estradiol and prolactin on sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels

Objective The present study investigated the sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels and the effects of thyroid-stimulating hormone(TSH),estradiol(E2)and prolactin(PRL)on NIS mRNA expression in mouse lactating mammary gland cells.

人溶菌酶cDNA的克隆及其在小鼠体内的表达

根据已发表的人溶菌酶 (human lysozyme,h L YZ) m RNA序列设计引物 ,以人乳腺第 1链 c DNA为模板 ,用 PCR扩增出 1.5 kb h L YZ双链 c DNA,其推导的氨基酸序列与国外发表的从胎盘、巨噬细胞和结肠中克隆的 h L YZ氨基酸序列同源性为 10 0 % ,与从人组织细胞中克隆的 h L YZ仅有 1个氨基酸差异 ,但与从中国人胎盘中克隆的 h L YZ具有6个氨基酸差异。将此 c DNA克隆入乳腺特异表达载体 ,用所获得的基因构件注射哺乳期小鼠 ,经 RT- PCR和微球菌溶解试验证明 ,上述基因构件能有效地驱动目的基因在小鼠乳腺中表达 ,表达量为 6 9.3mg/ L,表达具有较好的组织特异性。这些试验结果表明 ,本研究构建的表达 h L YZ c DNA的基因构件可以用于乳腺生物反应器的研制。

人凝血IX因子基因乳腺组织特异性表达载体的构建及其在奶山羊乳腺中的分泌性表达

人凝血ＩＸ因子基因乳腺组织特异性表达载体的构建及其在奶山羊乳腺中的分泌性表达张克忠卢大儒邱信芳（复旦大学遗传学研究所上海２００４３３）黄英黄淑帧（上海市儿童医院医学遗传研究所上海２０００４０）血友病Ｂ（ＨｅｍｏｐｈｉｌｉａＢ）是由于人凝血ＩＸ因子（ｈ...