转人溶菌酶基因小鼠乳腺生物反应器的建立

目的 研究人溶菌酶（human lysozyme，hLYZ）动物乳腺生物反应器制备的可行性。方法 将hLYZ基因与动物乳腺特异表达载体p205C3连接，所得重组载体p205C3-hLYZ用显微注射法建立转基因小鼠。结果 共出生了136只F0代小鼠，PER和Southern杂交检测基因整合阳性率分别为5．15％（2♀5♂）和2．94％（1♀3♂）。Western印迹检测结果表明，分泌在小鼠乳汁中的表达产物与正常hLYZ具有相同的分子量。目前转基因小鼠已经繁殖到B代，每一代基因整合阳性母鼠的乳腺均表达hLYZ，乳汁中的表达量最高达750mg/L。斑点杂交试验证明，表达有较强的组织特异性，除在乳腺表达外，仅在脾脏和小肠有一定的异位表达。结论 成功建立了hLYZ小鼠乳腺生物反应器。     

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.     

Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice

The expression of chimeric genes in the mammary gland of transgenic farm animals has become an alternative for the large-scale production of recombinant proteins and for the modification of milk composition. In this paper, we show that a mouse/human chimeric antibody against the human CD6 leukocyte antigen can be assembled and correctly folded by the mammary gland, and secreted to milk, where it maintains its specificity. The base sequences encoding for the heavy and light chain variable regions of the anti-CD6 mouse monoclonal antibody IOR-TI were cloned by the polymerase chain reaction from hybridoma cDNA, coupled to human heavy and light chain constant region genes, and inserted in a vector containing the 5′ regulatory region of the rabbit whey acidic protein gene. Transgenic mice were produced by conventional pronuclei microinjection techniques. Integration and transgene copy number were determined by Southern blot. Assembled human immunoglobulin was detected in milk using a sandwich ELISA. Expression levels of chimeric antibodies in milk were determined to be around 400 μg/ml by Western blot, using CHOderived chimeric IOR-T1 antibodies as reference. The chimeric antibodies produced in milk recognized human peripheral blood T lymphocytes by indirect immunofluorescence, with the classical patch-like pattern of IOR-T1.     

Gene expression analysis of immune-mediated arrest of tumorigenesis in a transgenic mouse model of HER-2/neu-positive basal-like mammary carcinoma

We previously showed that a vaccine combining interleukin 12 and allogeneic p185(neu)-positive mammary carcinoma cells completely prevented multifocal mammary carcinogenesis in HER-2/neu transgenic mice. To identify the molecular events responsible for effective tumor prevention and to define the tumor gene expression signature, we used microarrays to analyze the expression profile of mammary tissue of untreated transgenic mice and of vaccine-treated, tumor-free mice at different time points. Mammary tissue from vaccinated mice displayed a gene expression profile different from that of untreated, tumor-bearing mice but similar to that of normal/hyperplastic mammary gland. Comparison of treated and untreated mice at 15 weeks of age revealed up-regulation of genes encoding antibodies, chemokines, gamma-interferon-induced genes and inflammatory molecules, and down-regulation of early genes induced by tumor development. The gene expression signature of HER-2/neu-transformed tumor cells showed modulation of genes promoting proliferation, angiogenesis, migration, invasion, and metastasis and inhibiting apoptosis and immune response. Meta-analysis of microarray data on human breast cancer showed that the signature of tumors arising in murine HER-2/neu transgenic model correctly classified human HER-2/neu-expressing tumors and normal breast tissue. Moreover murine and human HER-2/neu-positive tumors share the signature of basal-like breast cancers. This gene expression analysis reveals the immune events associated with prevention of tumor development and shows that HER-2/neu transgenic mice represent a good model of a poor-prognosis group of human breast tumors.     

Mammary gland-specific secretion of biologically active immunosuppressive agent cytotoxic-T-lymphocyte antigen 4 human immunoglobulin fusion protein (CTLA4Ig) in milk by transgenesis

A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival. Tolerance induction is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side effects. The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient's immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical transplantation. To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals. Prior to the generation of transgenic farm animals, we tested our strategy in mice. Using the promoter of the sheep beta-lactoglobulin gene, we expressed our CTLA4Ig chimeric gene in the mammary gland of transgenic mice. The yield of CTLA4Ig was fivefold higher in transgenic milk than that from transfected cells. Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation. We showed that the production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that were fed with the transgenic milk. The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is feasible; further studies involving farm animals are warranted.     

A virus-neutralising antibody is not cytotoxic in vitro

Hybridoma cell lines are characterized by a preferential loss of the heavy chain gene. This observation has led to the theory that the immunoglobulin heavy chain possesses an intrinsic cytotoxic activity in some cell types. We have generated transgenic mice expressing the heavy and light chain genes of the virus-neutralising antibody A1 carrying constant domains of the human gamma1 and kappa isotype. Heavy chain and light chain transgenes were under trancriptional control of identical promoter regions derived from the mammary gland specific ovine beta-lactoglobulin gene. The copy number of the heavy chain transgene was consistently lower than the copy number of the light chain gene in all lines of transgenic mice. Moreover, the light chain gene was expressed in significant excess of the heavy chain gene in the lactating mammary gland in all transgenic lines. In several transgenic lines, the differences in antibody expression were greater than could be explained by the differences in transgene copy number. One potential cause of this phenotype could be a cytotoxic effect of free heavy chain protein in embryonic cells (resulting in differences in copy number) or mammary epithelial cells (resulting in differences in transgene expression). We therefore directly assessed the effect of the expression of free A1 heavy chain protein in epithelial cell lines and in murine embryonic stem cells. However, full-length A1 heavy chain mRNA and protein could be expressed transiently and stably in both epithelial and embryonic stem cells and had no detectable effect on cell viability. Taken together, these findings argue against an inherent cytotoxicity of the free A1 heavy chain protein in epithelial or embryonic cells.     

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.     

Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.     

Natural infection of swiss mice with Mouse Mammary Tumor Virus (MMTV): Viral expression in milk and transmission of infection

Summary Quantitative determinations of gp52, the main envelope glycoprotein, and p28, the main core protein, of MMTV, have been performed in about 1000 individual samples of milk of breeding females from our colony of MMTV-infected Swiss mice, a line characterized by a moderate incidence of mammary tumors. A computer analysis of the results showed: 1— an important individual variation, ranging from 0 to 120µg per ml of milk for p28, and from 0 to 320 µg per ml of milk for gp52; 2— a variation of the release of both antigens during a single lactation, with a maximum on the 7–8th day of nursing; 3— an increase of the release of both antigens with parity up to the 6th lactation, followed by a marked decrease during later lactations; 4— a higher degree of infection in the offspring of 2nd and 3rd litters. The possible dependence of viral expression and transmission of infection upon factors such as cyclic activity of the mammary gland and progressive immunization of mice against MMTV is analyzed. The status of our laboratory line of MMTV infected Swiss mice is discussed in comparison with high and low tumor incidence strains.With 4 Figures     

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-Itax gene

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of thelyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.The abbreviations used are ATL, adult T-cell leukemia; HTLV-I, human T-cell leukemia virus type I; IL-2, interleukin 2; IL-2R, interleukin 2 receptor; IL-6, interleukin 6; LTR, long terminal repeat.     

The housekeeping gene xanthine oxidoreductase is necessary for milk fat droplet enveloping and secretion: gene sharing in the lactating mammary gland

Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism occurring in most cell types. However, this housekeeping gene is expressed at very high levels in a number of mammalian tissues including the lactating mammary epithelium, suggesting additional roles for XOR in these tissues. Mice with targeted disruption of XOR were generated to assess these potential additional roles. XOR-/- mice are runted and do not live beyond 6 wk of age. Strikingly, however, XOR+/- females, although of healthy appearance and normal fertility, are unable to maintain lactation and their pups die of starvation 2 wk postpartum. Histological and whole-mount analyses showed that in XOR+/- females the mammary epithelium collapses, resulting in premature involution of the mammary gland. Electron microscopy showed that XOR is specifically required for enveloping milk fat droplets with the apical plasma membrane prior to secretion from the lactating mammary gland. We present evidence that XOR may have primarily a structural role, as a membrane-associated protein, in milk fat droplet secretion and thus XOR provides another example of "gene sharing". About 5% of women experience primary lactation insufficiency. The above observations suggest that human females suffering from xanthinuria, a deficiency in XOR, are potential candidates for lactation problems.     

Ha-ras and c-myc oncogene expression interferes with morphological and functional differentiation of mammary epithelial cells in single and double transgenic mice

We studied the effects of the tissue-specific and lactogenic hormone-dependent expression of the recombinant whey acidic protein (Wap)-ras and Wap-myc oncogenes on the differentiation of the mammary epithelium in transgenic mice. The histological appearance of mammary glands in pregnant, lactating, and postlactational animals and their ability to express milk protein genes were analyzed. Activated Ha-ras expression caused a decrease of milk protein synthesis during the lactation period. The formation of glandular epithelium and the postlactational regression of epithelial cells were not affected. c-myc expression impaired the development of the glandular epithelium, and milk protein synthesis was decreased strongly. Epithelial cell proliferation continued during lactation and postlactationally. Coexpression of both oncogenes in double transgenic mice synergistically affected differentiation and resulted in a high number of neoplastic foci. Palpable tumors were observed only after a latency of 3-4 months. Tumor cells utilize the Wap promoter hormone independently, express increased levels of Wap-ras and induce adjacent stromal cells to produce tenascin.     

Metabolism of immunoglobulin A in lactating mice: origins of immunoglobulin A in milk

The metabolism of albumin and IgA was studied in normal and lactating mice. Lactation resulted in significant changes in the metabolism of these proteins. The serum albumin concentration was lowered from 47 mg/ml in normals to 24 mg/ml in lactating mice. However, only a slight decrease in the serum concentration of IgA was observed during lactation. The proportion of polymeric and monomeric IgA in serum and milk was evaluated by gel exclusion chromatography. The onset of lactation led to a rise in the proportion of polymeric IgA (P-IgA) in serum from 37% to 51%. The proportion of P-IgA in milk was 65% and remained constant throughout lactation. P-IgA and albumin were shown to be efficiently transferred from the serum of lactating mice into their milk. Serum decay studies were performed to evaluate the turnover of the serum pools during lactation. The rates of disappearance from the serum of isotopically labeled albumin and P-IgA were observed to increase dramatically during lactation, suggesting that both of these two milk proteins might be derived at least in part from the serum. The sites of synthesis of milk IgA (local vs. extra-mammary gland) were evaluated by determining the extent of dilution of isotopically labeled serum IgA during transport through the mammary gland into the milk. Early in lactation, the majority of the IgA in mouse milk appeared to be derived from distant sites and transferred via the blood to the mammary gland. However, by day 8 of lactation, the isotopically labeled P-IgA in milk was significantly.diluted by the IgA synthesized in the mammary gland. Albumin and IgG were not diluted by local synthesis indicating that these proteins were exclusively serum-derived.     

Gene expression profiling: cell cycle deregulation and aneuploidy do not cause breast cancer formation in WAP-SVT/t transgenic animals

Microarray studies revealed that as a first hit the SV40 T/t antigen causes deregulation of 462 genes in mammary gland cells (ME cells) of WAP-SVT/t transgenic animals. The majority of deregulated genes are cell proliferation specific and Rb-E2F dependent, causing ME cell proliferation and gland hyperplasia but not breast cancer formation. In the breast tumor cells a further 207 genes are differentially expressed, most of them belonging to the cell communication category. In tissue culture breast tumor cells frequently switch off WAP-SVT/t transgene expression and regain the morphology and growth characteristics of normal ME cells, although the tumor-revertant cells are aneuploid and only 114 genes regain the expression level of normal ME cells. The profile of retransformants shows that only 38 deregulated genes are tumor-specific, and that none of them is considered to be a typical breast cancer gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Stromal Hoxa5 function controls the growth and differentiation of mammary alveolar epithelium.

Recent progress has enlightened the involvement of Hox genes in organogenesis. Several Hox genes are expressed in normal and neoplastic mammary glands. Using Hoxa5 mutant mice, we showed that Hoxa5-/- females present nursing defects. Characterization of the Hoxa5-/- mammary gland phenotype reveals changes in proliferation and differentiation of the epithelium of nulliparous and pregnant Hoxa5-/- females that precede the abnormal secretory activity at parturition. These defects likely underlie the incapacity of Hoxa5-/- dams to properly feed their pups. Hoxa5 expression is restricted to the mammary stroma at specific stages of mammary gland development. The loss of Hoxa5 function causes accelerated lobuloalveolar epithelium development, a phenotype that can be rescued upon grafting of mutant mammary epithelium into wild-type fat pads. Conversely, reciprocal grafting experiments demonstrate that Hoxa5-/- stroma cannot support normal proliferation of wild-type mammary epithelium. These data establish the essential contribution of Hoxa5 to mammary epithelium instruction by means of mesenchymal-epithelial crosstalk.