Mammary Gland Of Transgenic Mice Research Articles

The Expression of Recombinant Human Serum Albumin in the Mammary Gland of Transgenic Mice

AbstractHuman serum albumin (HSA) is widely used in the clinic for the treatment of several diseases in large amount each year. With the increasing demands of HSA in clinic and limited blood resource, recombinant HSA (rHSA) is becoming an attractive and alternative source for HSA production. In this study, we aimed to express rHSA in the mammary glands of transgenic mice by using a tissue-specific promoter and other regulatory elements. An rHSA expression vector was constructed bearing the cDNA and first intron of HSA under the control of bovine αs1-casein promoter with a 2 × chicken β-globin insulator in the front. Transgenic mice were generated and reverse transcription polymerase chain reaction showed that rHSA was expressed only in the mammary gland, indicating the tissue specificity of the bovine αs1-casein promoter in directing transgene transcription in transgenic mice. Enzyme-linked immunosorbent assay test showed that rHSA was successfully secreted into the milk of transgenic mice with the highest level at 1.98 ± 0.12 g/L. Our results indicate the ability of the bovine αs1-casein promoter to induce successful expression of rHSA in the mammary gland of transgenic mice.

Expression of functional plant sweet protein thaumatin II in the milk of transgenic mice

Thaumatin is a kind of natural sweet protein which has the characteristics of high sweetness, low calorie, safe and non-toxic, etc. And it has high commercial value in food processing, medicine and other fields.In this study, we employed a sequence that encode thaumatin II, along with goat beta-casein 5′ and 3′ regulatory elements, to construct a mammary gland-specific expression vector. Transgenic mice were generated using pronuclear microinjection, and the expression of thaumatin and its biological activities were assayed in the milk.PCR and southern blot analysis confirmed that the mice harbor thaumatin II transgenes. Thaumatin II retaining their sweetness was expressed in the mammary glands of transgenic mice, as determined by ELISA, western blot and sweetness intensity test.This research describes an initial step in the production of plant protein thaumatin-sweetened milk from large animals such as cow and goat.

Expression of a recombinant anti-programed cell death 1 antibody in the mammary gland of transgenic mice

Nivolumab, a fully human IgG4 anti-programed cell death 1（PD-1）antibody, is recently one of the most popular and successful therapeutic monoclonal antibodies in clinical use. With the increasing demands for Nivolumab and other therapeutic monoclonal antibodies, the mammary gland bioreactor has been regarded as another choice for the production of recombinant monoclonal antibodies besides mammalian cell culture. Here, we expressed a recombinant human anti-PD-1 antibody in the mammary glands of transgenic mice. Two expression vectors were constructed bearing the heavy and light chains of anti-PD-1 antibody respectively under the control of bovine αs1-casein promoter. Transgenic mice were then generated by co-microinjection of the two expression cassettes. Three F0 founders with both heavy chain and light chain positive were obtained. Transgenes of both chains were detected to be stably transmitted to the offspring. The recombinant antibody was detected in the milk of transgenic mice with the highest expression level up to 80.52 ± 0.82 mg/L and could specifically binds to the human PD-1 antigen. Therefore, our results suggest the feasibility of anti-PD-1 antibody production in the milk of transgenic animals.

H2AX Promoter Demethylation at Specific Sites Plays a Role in STAT5-Induced Tumorigenesis.

Deregulated STAT5 activity in the mammary gland of transgenic mice results in parity-dependent latent tumorigenesis. The trigger for cell transformation was previously associated with hyperactivation of the H2AX proximal promoter in a small basal cell population during pregnancy. The current study focuses on the latent activation of tumor development. H2AX was highly expressed in carcinoma and adenocarcinoma as compared to the multiparous mammary gland, whereas pSTAT5 expression decreased in a tumor type-dependent manner. In contrast to the pregnant gland, no positive correlation between H2AX and pSTAT5 expression could be defined in carcinoma and adenocarcinoma. Using targeted methylation analysis, the methylation profile of the H2AX promoter was characterized in the intact gland and tumors. Average H2AX promoter methylation in the tumors was relatively high (~90%), but did not exceed that of the multiparous gland; 5mC methylation was higher in the differentiated tumors and negatively correlated with its oxidative product 5hmC and H2AX expression. Individual analysis of 25 H2AX promoter-methylation sites revealed two consecutive CpGs at positions -77 and - 54 that were actively demethylated in the multiparous gland, but not in their age-matched virgin counterpart. The different methylation profiles at these sites distinguished tumor types and may assume a prognostic role. In-silico and ChIP analyses revealed overlapping methylation-independent SP1-binding and methylation-dependent p53-binding to these sites. We propose that interference with SP1-assisted p53-binding to these sites abrogates H2AX's ability to arrest the cell cycle upon DNA damage, and contributes to triggering latent development of STAT5-induced tumors in estrapausal multiparous mice.

Production of biologically active human factor IX-Fc fusion protein in the milk of transgenic mice.

To investigate the feasibility of producing human IgG1 Fc fragment fused factor IX (FIX-Fc) in the milk of transgenic animals, for an alternative possible solution to the unmet need of FIX-Fc products for hemophilia B treatment. Six founder lines of transgenic mice harboring FIX-Fc cassette designed to be expressed specifically in the mammary gland were generated. FIX-Fc protein was secreted into the milk of transgenic mice with preserved biological activity (with the highest value of 6.2IU/mL), similar to that of the non-fused FIX transgenic milk. RT-PCR and immunofluorescence analysis confirmed that FIX-Fc was specifically expressed in the mammary gland. The blood FIX clotting activities were unchanged, and no apparent health defects were observed in the transgenic mice. Moreover, the stability of FIX protein in milk was increased by the Fc fusion. It is feasible to produce biologically functional FIX-Fc in the mammary gland of transgenic mice. Our preliminary results provide a foundation for the potential scale-up production of FIX-Fc in the milk of dairy animals.

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice.

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1β. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.

Transcription termination sequences support the expression of transgene product secreted with milk.

Expression of the reporter gene in transgenic animals depends on the surrounding chromatin environment. Recent genome-wide studies have shown that, in mammals, the entire genome is transcribed. Transcription through a transgene often has a negative effect on the expression of a reporter gene. Here, we compared the ability of well-studied chicken chromatin insulator HS4 and bidirectional transcription terminators from the human genome to support high-level expression of the firefly luciferase gene (Fluc) under control of the previously characterized goat β-casein gene promoter. The insertion of HS4 or either of the two transcription terminators upstream of the promoter resulted in tenfold enhancement of Fluc expression in the mammary glands of transgenic mice. These results suggest that transcriptional terminators, similar to the HS4 insulator, can be used to improve the reporter gene expression in transgenic animals.

Abstract IA14: Breaking bad: Cancer-associated fibroblasts are reprogrammed from growth inhibitory to pro-inflammatory and tumor promoting in breast cancer

Abstract Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. We previously demonstrated that Cancer-Associated Fibroblasts (CAFs) orchestrate tumor-enhancing inflammation in multiple mouse and human malignancies, including breast cancer. Although breast cancer is one of the major tumor types where CAFs were shown to be tumor promoting, there is no detailed analysis of the dynamic changes in CAFs characteristics and function in correlation with tumor progression. We therefore set out to characterize the dynamic changes in CAFs during progression of mammary carcinogenesis, in a transgenic mouse model of human breast cancer. We profiled the transcriptome of fibroblasts isolated from normal mammary glands, or from mammary glands of transgenic mice at defined tumorigenic stages. Data analysis revealed unique CAF gene signatures that correspond to distinct tumor stages, with only partial overlap between the stages, suggesting co-evolution of the microenvironment with tumorigenic progression. Interestingly, the gene signature of fibroblasts isolated from hyperplastic lesions is inverse to that in CAFs from neoplastic stages, and has a growth inhibitory phenotype, while CAFs from mammary tumors express pro-inflammatory and tumor promoting gene signatures. Moreover, we functionally demonstrate that CAFs isolated from late carcinoma express a gene signature that links tissue damage with tumor-promoting inflammation. To identify tumor-secreted factors capable of reprogramming normal mammary fibroblasts to pro-inflammatory CAFs, we performed a proteomic screen of breast cancer cells secretome, and identified Osteopontin (OPN). Knockdown of OPN in tumor cells in vivo resulted in attenuated stromal activation, reduced immune cell recruitment and inhibition of tumor growth. Taken together, our findings reveal reciprocal crosstalk between fibroblasts and cancer cells which drives CAF reprogramming, to facilitate tumor progression in breast cancer. Citation Format: Yoray Sharon, Nour Ershaid, Yael Raz, Noam Cohen, Metsada Pasmanik-Chor, Tamar Geiger, Neta Erez. Breaking bad: Cancer-associated fibroblasts are reprogrammed from growth inhibitory to pro-inflammatory and tumor promoting in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA14.

The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment.

Amplification and over expression of erbB2/neu proto-oncogene is observed in 20–30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 due to constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2Ex16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16 derived mammary tumors exhibit several unique features that distinguish it from the conventional ErbB2 models expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2 derived tumors that express luminal keratins, ErbB2ΔEx16 derived tumors exhibit high degree of intra-tumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibited distinct signaling and gene expression profiles that correlated with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland ‘bioreactor’ is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P < 0.05). While hFVIIIBD was present in multiple organs in CMV-hFVIIIBD mice, P1A3-hFVIIIBD mice showed negligible hFVIIIBD expression in organs other than the mammary glands. This study demonstrated that the mammary gland-specific P1A3-hFVIIIBD vector was more suitable for the generation of hFVIIIBD mammary gland bioreactor.

Investigation of hFVIII production in mammary glands of transgenic mice.

Hemophilia A is an X-linked disorder affecting 1 in 10,000 males. The disease is caused by a defect or mutation of factor 8 or 9. Human factor 8 gene (hFVIII) is a relatively large gene consisting of 26 exons and approximately 2,351 amino acids with a length of 9 Kb mRNA. Expression of hFVIII in mammalian milk is becoming a widespread strategy for high-level production of hFVIII because of the most complex post-translational modifications. The aim of this study was the cloning and expression of hFVIII in mammary glands of two transgenic mice. To obtain a recombinant plasmid, first a plasmid carrying an FVIII gene fragment (pCMV6-hFVIII) was digested by EcoRI-SalI restriction enzymes and then the fragment was purified from agarose gel and inserted into a pUCWAP7 vector carrying a tissue-specific promoter (mWAP 4.1 kbp). After that, it was isolated by agarose gel and transferred into the murine zygotes by standard microinjection methods. Methods for expression of recombinant FVIII RT-PCR and ELISA were studied. The results show the successful expression of factor FVIII gene and its product in the mouse mammary glands.

Mammary gland-specific expression of biologically active human osteoprotegerin in transgenic mice.

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat β-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 µg/ml. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

Abstract B103: Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis

Abstract Cancer-associated fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. We demonstrated that CAFs also mediate tumor-enhancing inflammation: CAFs isolated from pre-neoplastic and tumor tissue expressed a pro-inflammatory gene signature and functioned to recruit macrophages and enhance tumor angiogenesis in a mouse model of squamous carcinoma. Moreover, we demonstrated that pro-inflammatory signaling by CAFs is operative also in mouse and human breast carcinogenesis. Although breast cancer is one of the major tumor types where CAFs were shown to be tumor promoting, there is no detailed analysis of CAF characteristics and function in correlation with tumor progression. We therefore set out to characterize the dynamic changes in CAFs during progression of mammary carcinogenesis, in a transgenic mouse model of human breast cancer, the MMTV-PyMT mice. We show that normal mammary fibroblasts can be “educated” by mammary carcinoma cells to become activated, pro-inflammatory CAFs. Furthermore, transcriptome profiling of fibroblasts isolated from normal mammary glands, or from mammary glands of transgenic mice at defined tumorigenic stages (hyperplasia, early carcinoma, invasive adenocarcinoma) revealed distinct CAF gene expression signatures that correspond to different tumor stages, with only partial overlap between the stages, suggesting co-evolution of the microenvironment with tumorigenic progression. Interestingly, the gene signature of fibroblasts isolated from hyperplastic lesions is inverse to that in CAFs from neoplastic stages, and has a putative growth inhibitory phenotype, while CAFs from early and late carcinoma express pro-inflammatory and tumor promoting gene signatures. Elucidating the molecular mechanisms by which CAFs mediate cancer-related inflammation in breast cancer will greatly increase our understanding of stromal pathways that contribute to tumorigenesis, in an effort to identify novel stromal molecular targets for combinatorial therapeutic approaches to combat breast cancer progression. Citation Format: Yoray Sharon, Yael Raz1,2, Lina Alon, Neta Erez. Proinflammatory signaling by cancer-associated fibroblasts co-evolves during mammary carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B103.

Effect of annatto-tocotrienols supplementation on the Development of Mammary Tumors in HER-2/neu Transgenic Mice

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

Expression of biologically active human interferon alpha 2b in the milk of transgenic mice

Interferon alpha 2b (IFNα-2b) is an important immune regulator widely used in clinic, for the treatment of chronic hepatitis, hairy cell leukemia, chronic myelogenous leukemia and multiple myeloma, etc. The clinically used IFNα-2b is generally produced by E.Coli, which lacks the post-translational O-glycosylation presents on naturally synthesized protein, and has a short serum half-life. In this study, a transgenic cassette pBCN-IFN-pA-CMV-EGFP was constructed, with a 5.2kb beta-casein regulation fragment from Jersey cow and a 6×His tagged human Interferon alpha 2b (hIFNα-2b) gene fragment. By using pronuclear microinjection technique, transgenic mice were generated and the expression of IFNα-2b in the milk was assayed. The hIFNα-2b was correctly translated in milk of transgenic mice according to Western blot analysis. The expression level of hIFNα-2b was varied among the transgenic mice, and the highest one was about 29.71μg/L. The recombinant protein exhibited biological activity in vitro by increasing the luminescence value and the MxA gene expression in established WISH cells, and the specific activity is approximately 2.8×10(7)IU/mg. The expression of recombinant hIFNα-2b in mammary glands of transgenic mice constitutes an important step towards low-cost and full biological activity production of this protein drug in mammary gland bioreactor.

On How Mammary Gland Reprogramming Metalloproteinases Couple Form with Function

On How Mammary Gland Reprogramming Metalloproteinases Couple Form with Function

The Construction and Expression of Lysine-Rich Gene in the Mammary Gland of Transgenic Mice

Lysine is the limiting amino acid in cereal grains, which represent a major source of human food and animal feed worldwide, and is considered the most important of the essential amino acids. In this study, β-casein, αS2-casein, and lactotransferrin cDNA clone fragments encoding lysine-rich peptides were fused together to generate a lysine-rich (LR) gene and the mammary gland-specific expression vector pBC1-LR-NEO(r) was constructed. Transgenic mice were generated by pronuclear microinjection of the linearized expression vectors harboring the LR transgene. The transgenic mice and their offspring were examined using multiplex polymerase chain reaction (PCR), Southern blotting, reverse transcriptase-PCR, in situ hybridization, and Western blotting techniques. Our results showed that the LR gene was successfully integrated into the mouse genome and was transmitted stably. The specific LR gene expression was restricted to the mammary gland, active alveoli of the transgenic female mice during lactation. The lysine level of the two transgenic lines was significantly higher than that of nontransgenic controls (p<0.05). In addition, the growth performance of transgenic pups was enhanced by directly feeding them the LR protein-enriched transgenic milk. Our results demonstrated that lysine-rich gene was successfully constructed and expressed in mammary gland of transgenic mice. This study will provide a better understanding of how mammary gland expression systems that increase the lysine content of milk can be applied to other mammals, such as cows.

Igf1r is not required for AIB1-induced mammary hyperplasia and ductal branching

The oncogene AIB1 (amplified in breast cancer 1) is a transcriptional coactivator which is up-regulated in many types of tumors including breast cancer. Studies with cell lines and animal models reveal that AIB1 interacts with the IGF-I signaling pathway at different molecular levels. To determine whether AIB1-dependent cell growth requires IGF-I signaling, we deleted the Igf1r gene specifically in the mammary gland of transgenic mice which overexpress AIB1 and are characterized by the development of epithelial hyperplasia, a pre-neoplastic change in breast tissue. Loss of Igf1r alone reduced cell proliferation, ductal branching and fat pad occupancy in comparison with wild-type glands. However, in the transgenic mice that overexpress moderate levels of AIB1, the absence of Igf1r had a minimal effect on epithelial hyperplasia and ductal branching in the mammary gland. Thus, our results confirm the essential role of Igf1r in mammary gland morphogenesis and demonstrate that overexpression of AIB1 circumvents the requirement for the Igf1r pathway in promoting epithelial growth during mammary development.