乳腺高效表达人溶菌酶转基因小鼠的制备

目的　研究自行构建的动物乳腺特异表达载体p2 0 5C3的表达特性。方法　将人溶菌酶 (hLYZ)cDNA插入p2 0 5C3载体 ,用获得的基因构件注射小鼠受精卵 ,用PCR和Southernblot对出生鼠进行基因整合检测 ,用微球菌溶解试验和Westernblot对表达产物进行鉴定。结果　共出生 136只F0 代小鼠 ,从中筛选出 4只 (1♀ 3♂ )转基因整合阳性鼠 ,其中的 1只母鼠不表达hLYZ ,1只雄鼠的 4只F1 代母鼠乳汁中hLYZ的表达量分别为 5、75、175和 2 0 0 μg ml,纯合后的 3只F3代母鼠乳汁中hLYZ的表达量分别为 5 2 6 μg ml、6 4 8μg ml和 75 0 μg ml,表达仅限于乳腺中 ,表达产物的相对分子质量与正常hLYZ相同。结论　p2 0 5C3能驱动hLYZcDNA在小鼠乳腺中特异和高效表达 ,可以用于动物乳腺生物反应器的研制。     

人Man2c1转基因小鼠模型的建立

目的 为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法 构建pIRKS2-EGFP-hMan2c1重组表达载体，经体外转染实验鉴定转染的基因能在COS-7细胞表达后，注射人ICR小鼠受精卵，以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Westernblot分析hMan2c1在转基因小鼠的表达。结果 在116只原代小鼠中，有7只hMan2c1基因组PCR阳性。在所检测的20只F1代小鼠中，有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中，有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达，确定基因组PCR阳性的7个系中有4个系阳性。结论 建立了4个稳定表达hMan2c1的转基因小鼠系，为深入研究MAN2C1的生物学意义打下了基础。     

应用显微注射法建立转基因小鼠

应用显微注射法建立转基因小鼠谢卫兵陈汉源曾位森罗琛（第一军医大学组胚教研室广州５１０５１５）转基因动物是研究基因表达调控及表达产物生物学效应的最佳体系之一，同时作为一种新型的生物反应器，在医学、农、林、畜牧业等领域有着广泛的应用前景。目前采用基因转移...     

人癌胚抗原相关细胞黏附分子1介导的小鼠淋病奈瑟菌易感性

目的 研究制备淋病奈瑟菌感染人癌胚抗原相关细胞黏附分子1(hCEACAM1)转基因小鼠模型的可行性.方法 用hCEACAM1真核表达载体pCDPGICEA1,通过显微注射法制备转基因小鼠,PCR及序列分析法检测目的基因在小鼠基因组中的整合,Western blot及FACS技术检测目的基因的表达,镜检及培养法检查淋病奈瑟菌对转基因小鼠的感染.结果 产生的22只F0代小鼠中4只为转基因整合阳性,其中1只可表达hCEACAM1蛋白,且目的蛋白表达在细胞膜上.淋病奈瑟菌可在转基因小鼠中形成感染.结论 hCEACAM1转基因小鼠可作为淋病奈瑟菌感染研究的转基因动物模型.     

MagA转基因小鼠模型的建立

目的建立MagA转基因小鼠,为活体MRI成像系统提供重要的实验动物模型。方法采用显微注射法．将MagA基因导入小鼠受精卵,再培育成转基因小鼠并繁殖传代。利用PCR方法鉴定转基因首建鼠及其子代基因型。结果酶切和DNA测序分析证实MagA基因准确克隆表达载体设计位点,目的基因序列与GenBank中MagA序列完全一致。通过PCR分析,进行转基因整合检测。目前转基因小鼠已稳定传代至第5代。结论MagA基因在转基因小鼠中整合,成功建立了MagA转基因小鼠。MagA转基因小鼠将成为MRI影像系统的重要实验动物模型。     

HBD-2转基因小鼠的建立及鉴定

人β防御素2（Human beta defensin 2，HBD-2）是人体抗菌肽的重要分子，体外试验研究证明具有广谱抗微生物活性，为开展其在整体水平上的功能研究，建立HBD-2转基因小鼠模型。用分子克隆方法构建了带CMV启动子的HBD-2全长cDNA真核重组表达质粒pCDNA3．1-HBD2，以此为摸板，PCR扩增出带CMV启动子和BGH polyA尾的HBD-2基因片段，用显微注射技术将此微基因导入小鼠雄原核中，于M16培养液培养后经输卵管移植到受体鼠体内让其怀孕产生子代小鼠。PCR法检测外源基因在小鼠基因组中的整合，RT—PCR和免疫组化法检测HBD-2在小鼠组织的表达。PCR检测结果显示17只F1代转基因鼠中4只检测到阳性信号，RT—PCR和免疫组化检测显示HBD-2在其F1代转基因小鼠生殖道、呼吸道、泌尿道以及血管内皮等组织部位广泛表达。实验表明HBIN2基因已整合到小鼠基因组且广泛表达，为进一步研究HBD-2的生物学功能及基因调控提供了有用的整体动物模型。     

携带淀粉样前体蛋白瑞典型突变基因转基因小鼠的制备及体内表达研究

目的 建立携带人类淀粉样前体蛋白瑞典型突变（Swedish mutation of amyloid precursor protein,APPSWE)基因的转基因小鼠模型。方法 采用受精卵原核显微注射法，将人类APPSWE转基因导入C57及昆明种小鼠受精卵内，然后将注射后保持完整的受精卵移植到假孕母鼠的输卵管内，然后应用聚合酶链反应（polymerase chain reaction,PCR)、荧光原位杂交及逆转录PCR分析子代小鼠中外源基因的整合及表达情况。结果 注射后卵的存活率和幼子出现率分别为76.62%和10.38%；外源基因整合率为35.29%；共获得6只首建小鼠，已稳定传3代，PCR检测共55只阳性；提取阳性小鼠心、脑、肝、肾组织及骨骼肌以人类APPSWE基因外显子特异的引物进行逆转录PCR分析，结果发现其心、脑组织及骨骼肌中具有人类APPSWE基因的表达。结论 说明携带淀粉样前体蛋白瑞典型突变基因的转基因小鼠模型已制备成功。     

人血小板生成素基因乳腺特异表达载体的构建和表达

目的为了获得能在转基因动物乳汁中高效表达人血小板生成素(TPO)的特异表达载体。方法将山羊β-乳球蛋白基因5′端上游-3.9kb和-1.9kb调控序列及β-酪蛋白启动子分别与人TPO基因连接,构建了pB3.9T、pB1.9T和pPT3个乳腺特异表达载体。将上述载体分别进行瞬间转染和稳定整合筛选实验,从mRNA及蛋白水平检测TPO基因的表达。结果BLG3.9、BLG1.9和P1A33种调控元件都可以启动人TPO在乳腺细胞中特异表达。瞬时转染时3种载体表达水平依次为pPT>pB3.9T>pB1.9T。但当外源基因稳定整合入细胞基因组后,TPO表达量持续升高,且pB3.9T表达量明显超过另外两种载体。结论证实了BLG转录子去阻遏和激活转变调节的存在,可能在其5′端-3.9kb与-1.9kb之间存在这一调节区域。并且与BLG1.9相比,BLG3.9能更有效地启动人TPO基因在乳腺细胞中的表达,也说明在-3.9至-1.9kb之间可能存在特殊的增强序列。     

乳腺生物反应器的基础研究——载体构建,检测及反应器的建…

乳腺生物反应器的研究和开发是近年生物工程领域的研究热点。乳腺特异性表达调控的研究和表达载体的构建是该工作的基础和关键。本文介绍了乳腺蛋白基因表达调控的研究现状，外源基因乳腺特异性表达载体构建策略以及乳腺生物反应器建立方法的研究进展。     

Efficient human IFN-gamma expression in the mammary gland of transgenic mice.

Two hybrid genes (BLG-HuIFN-gamma2 and BLG-HuIFN-gamma3) were constructed on the basis of sheep beta-lactoglobulin (BLG) and human interferon-gamma (HuIFN-gamma) gene sequences. They were used to direct HuIFN-gamma synthesis in the mammary gland of transgenic mice. HuIFN-gamma was efficiently produced in the mammary gland of transgenic mice. BLG-HuIFN-gamma2 transgenic females expressed HuIFN-gamma in the milk at concentrations up to 570 mg/ml, and BLG-HuIFN-gamma3 transgenic females expressed up to 350 mg/ml. All females carrying the BLG-HuIFN-gamma3 gene expressed HuIFN-gamma in their milk. No significant changes were observed in the HuIFN-gamma expression level during the lactation period. Using RT-PCR analysis, ectopic expression for both hybrid genes was found in transgenic mice. Despite ectopic expression of HuIFN-gamma in transgenic mice, their development and pregnancy were normal. The heritability of the HuIFN-gamma expression level in milk was demonstrated up to the F2 generation. This work demonstrates that hybrid genes have the potential to develop in transgenic domestic animals producing HuIFN-gamma in milk.     

Targeted expression of activated Rac3 in mammary epithelium leads to defective postlactational involution and benign mammary gland lesions

Rac3, a novel member of the Rho subfamily of the small GTPases, is frequently activated in cultured breast cancer cells and has been shown to mediate its effect via the p21-activated kinase (Pak) pathway. In order to evaluate these findings in vivo, we generated transgenic mice that express human constitutively active V12Rac under the control of the mouse mammary tumor virus (MMTV) promoter, which targets the transgene expression to the mammary epithelium. V12Rac3 expression could be detected during the first pregnancy, and the transgenic mammary gland tissues displayed an elevated Pak1 phosphorylation. Although milk proteins, beta-casein and whey acidic protein were expressed and milk fat globules accumulated normally during pregnancy, 60% of transgenic mothers failed to nurse their pups. Surprisingly, although full lactational differentiation was never achieved in transgenic mice, gland involution was incomplete. For 5 days after weaning, involution was normal, but thereafter, epithelial islands characteristic of this early stage of involution persisted for months. The apoptotic index decreased after 5 days, and these glands were associated with increased p38 MAPK phosphorylation. Nine months postpartum, the transgenic mammary glands still demonstrated a large amount of persistent epithelial islands and abnormally large ducts with lymphocyte infiltration, whereas the tissues of non-transgenic controls had returned to their normal 'virgin-like' phenotype. These data show that sustained activation of Rac3 in the mammary epithelium leads to impaired mammary gland physiology and results in the formation of mammary gland lesions.     

Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells

The present study is undertaken to investigate the immune response that was induced by the recombinant spike (S) protein from swine-transmissible gastroenteritis virus (TGEV) expressed in mouse mammary cells. A mammary-specific expression vector pEBS containing the full-length cDNA of S gene was constructed and expressed in the mouse mammary cells (EMT6). The recombinant S protein from culture supernatant of transgenic EMT6 was harvested and immunized BALB/c mice. The results demonstrated recombinant S protein was expressed at high levels in mammary cells by Western blotting and enzyme-linked immunosorbent assay (ELISA) detection. The antibody titer in BALB/c mice following immunization with recombinant S protein was detectable after the first immunization. Maximum titers of antibody (8.86+/-0.19 ng/ml of serum) were attained after the second immunization. In conclusion, the recombinant S protein expressed in mammary cells was able to elicit substantial immunological response against TGEV. This lays the basis for using mammary gland bioreactor generating edible vaccine.     

In vivo MRI of early stage mammary cancers and the normal mouse mammary gland

Since the advent of screening mammography, approximately one-quarter of newly diagnosed breast cancers are at the earliest preinvasive stage of ductal carcinoma in situ (DCIS). Concomitant with this improvement in early detection has been a growing clinical concern that distinguishing aggressive from indolent DCIS is necessary to optimize patient management. Genetically engineered mouse models offer an appealing experimental framework in which to investigate factors that influence and predict progression of preinvasive neoplasias. Because of the small size of early stage carcinomas in mice, high-resolution imaging techniques are required to effectively observe longitudinal progression. The purpose of the present study was to evaluate the feasibility of MRI for assessment of in situ mammary neoplasias and early invasive mammary cancers that stochastically arise in mammary glands of C3(1) SV40 Tag transgenic mice. Additionally, images of normal mammary glands from wild-type FVB/N mice were acquired and compared with those from transgenic mice. Sixteen mice underwent MR examinations employing axial two-dimensional multi-slice gradient recalled echo scans (TR/TE =～1000/5.5 ms) with fat suppression in a two-step process targeting both the upper and lower mammary glands. MRI successfully detected in situ and early invasive neoplasias in transgenic mice with high sensitivity and specificity. The average signal-to-noise ratio (SNR) of in situ lesions on fat-suppressed high-resolution T(1) -weighted images was 22.9, which was lower than that of invasive tumors, lymph nodes and muscle (average SNR of 29.5-34.9, p < 0.0001) but significantly higher than that of normal mammary tissue (average SNR = 5.5, p < 0.0001). Evaluation of wild-type mammary glands revealed no cancerous or benign lesions, and comparable image contrast characteristics (average SNR = 5.2) as compared with normal tissue areas of transgenic mice. This present study demonstrates that MRI is an excellent candidate for performing longitudinal assessment of early stage mammary cancer disease progression and response to therapy in the transgenic model system.     

Impaired lactation in mice expressing dominant‐negative FADD in mammary epithelium

The Fas‐associated death domain (FADD/Mort1) adaptor protein was originally identified as a key mediator of apoptosis, although pleiotropic functions for FADD have also been reported. FADD‐mediated tumoricidal effects have been described in breast cancer cells; however, its physiological role in normal mammary gland epithelium is not well understood. To determine the role of FADD signaling during mammary gland development, we generated transgenic mice overexpressing dominant‐negative FADD (DN‐FADD) in mammary epithelium, using the steroid responsive mouse mammary tumor virus promoter. Transgenic mice exhibited a perturbation in lactation resulting in impaired milk production and pup growth retardation. Reduced expansion of alveoli was evident during early lactation with extensive shedding of luminal alveolar cells. Significantly more TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end‐labeling)‐positive cells were present at this time point and a subsequent increase in bromodeoxyuridine‐positive cells was observed. These findings suggest a role for FADD in maintaining the survival of mammary secretory alveolar cells after the establishment of lactation. Developmental Dynamics 238:1010–1016, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of mouse mammary tumor virus envelope protein does not prevent superinfection in vivo or in vitro.

Inbred mice expressing endogenous mouse mammary tumor virus envelope proteins can be infected with exogenous virus, and the mammary tumors that develop in these mice usually have many proviruses integrated in their genomes, indicating that this virus is not subject to receptor interference. We show here that transgenic mice expressing an exogenous mouse mammary tumor virus (C3H) envelope protein can still be infected with this virus. Moreover, cultured mammary gland cells expressing the mouse mammary tumor virus (C3H) envelope protein can be superinfected with pseudotyped viruses bearing that same protein. Thus cellular expression of the mouse mammary tumor virus envelope protein does not block superinfection in vivo or in vitro.     

Diet‐induced obesity disrupts ductal development in the mammary glands of nonpregnant mice

Mammary glands develop postnatally in response to the hypothalamic‐pituitary‐gonadal axis. Obesity‐induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high‐fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin. Developmental Dynamics 238:1092–1099, 2009. © 2009 Wiley‐Liss, Inc.     

不同类型乳腺增生性疾病患者心理健康的比较

目的 探讨不同类型乳腺增生性疾病患者的心理健康状况.方法 应用HP Agilent IMAGE POINTHX彩超仪,诊断和区分单纯性乳腺增生症(研究1组)、乳腺囊性增生病(研究2组)和健康妇女各60例,在月经周期的月经期、增生期、分泌期、月经前期采用SCL-90评定精神状态.结果 两研究组在月经期和月经前期躯体化、人际敏感、抑郁、焦虑等因子分比健康对照组高;在增生期两研究组抑郁、焦虑等因子分比健康对照组高,研究l组的躯体化因子分比健康对照组高;在分泌期两研究组躯体化、抑郁、焦虑等因子分比健康对照组高;月经期研究1组躯体化、焦虑因子分及月经前期焦虑因子分比研究2组高,有显著性差异(P＜0.05),其他因子两组问差异不显著.结论 乳腺增生性疾病患者心理健康状况较健康妇女差,主要表现为躯体化、人际敏感、抑郁、焦虑,并随月经周期而变化,而且单纯性乳腺增生症患者心理健康状况较乳腺囊性增生病患者更差.     

Sinus‐like dilatations of the mammary milk ducts,Ki67 expression,and CD3‐positive T lymphocyte infiltration,in the mammary gland of wild European rabbits during pregnancy and lactation

Sinus‐like dilatations of the mammary duct are recognisable in the mammary gland of pregnant and lactating wild European rabbits. These dilatations exhibit a bilaminar epithelial lining, with luminal epithelial cells expressing basal and lateral E‐cadherin. Occasional binucleated mammary epithelial cells are present in the luminal layer. Underlying the luminal epithelial cells is a basal layer of cytokeratin 14‐positive cells, supported by a thin layer of fibrous tissue. Multi‐segmental epithelial proliferation, as indicated by Ki67 expression, is apparent in the luminal epithelial cells, suggesting a capacity for division during pregnancy and lactation. CD3‐positive T lymphocytes are present both intraepithelially, suggesting exocytosis, and in foci subjacent to the ductular epithelium. We consider that sinus‐like dilatations of the mammary duct may have the potential to give rise to a subset of the mammary gland neoplasms classified as ductal in origin. Milk accumulation in these sinus‐like dilatations is likely to provide a niche for bacterial replication in cases of mastitis in rabbits. These structures are an important component of the innate immune system of the mammary gland, both as a physical barrier and as an interface between the milk and mammary immune cells.