The study of mammary gland bioreactor is in the ascendant. In order to generate transgenic goats of well-controlled expression of exogenic genes, we constructed a human lactoferrin (hLF) gene targeting vector containing promoter, exon 1, intron1 and some of exon 2 (about 6.1 kb fragment) and exon 6 approximately 9 (about 3.3 kb fragment) of the goat beta-casein gene as well as hLF minigene, neo gene inserted into them and tk gene ligated to the 3' end of the construct. The 9.4 kb goat genomic sequences as homologous arms were initially amplified by PCR with local goat tissue DNA. The expression vector was named pBC-tk-neo-hlf. Then the recombinant plasmid pBC-tk-neo-hlf containing hLF minigene was transfected into mice mammary tumor cell line C127 by liposome, cell clones were selected with G418. After proliferating, the transfected cells were induced with insulin, luteotropic hormone and hydrocortisone. The result of Western-blotting analysis showed that the transfected cells can secrete hLF protein, and the recombinant protein expressed in cultured cell supernatant has the similar molecular weight as the native protein. The expression level detected by ELISA was 0.21 microg/mL. This result indicated that the targeting vector could efficiently direct the expression of hLF in mammary cells,and it confirmed the validity of the constructed vector. At the same time, C127 cell line proved to be useful for evaluating the regulation of a foreign gene expression in mammary gland specific expression vector.
Animals ; Caseins ; genetics ; Cell Line, Tumor ; Goats ; Humans ; Lactoferrin ; genetics ; Mammary Glands, Animal ; cytology ; metabolism ; Mice ; Molecular Weight ; Transfection

The aim of the study was to construct a recombinant adenovirus overexpression vector for Sterol Regulatory Element Binding Protein-1 (SREBP-1) of Xinong Saanen dairy goat, and to detect its effect on genes related to fatty acid metabolism in goat mammary epithelial cells, to establish foundation for further study of its roles in metabolism of fatty acid synthesis and lactation. First, we designed primers based on the SREBP-1 gene sequence in GenBank for PCR amplification and inserted the sequence into shuttle vector pAdTrack-CMV. The recombinant plasmid pAdTrack-CMV-SREBP-1 linearized by Pme I was transformed into E. coli BJ5183 competence cell containing the backbone vector pAdEasy-1 to obtain recombinant vector pAd-SREBP-1 by homologous recombination. pAd-SREBP-1 was linearized by Pac I and transfected into HEK 293 cell. Then we infected goat mammary epithelial cells with recombinant adenovirus which was packaged in HEK 293 cell line. The results showed that the recombinant adenovirus vector containing SREBP-1 was successfully constructed, and the titer of virus was 10(9) U/mL. Compared with the control group, mRNA level of SREBP-1 increased by about 15 times after infected for 48 h and 30 times after infected for 72 h. Fatty acid synthase (FASN) and Acetyl-CoA carboxylase (ACC) was upregulated by almost 2 times. The expression level of Peroxisome proliferator activated receptorgamma (PPARgamma) increased by 1.5 times. Liver X receptoralpha (LXRalpha) and Adipose triglyceride lipase (ATGL) upregulated by 1.2 times compared with that of control. But Stearoyl-coenzyme A desaturase (SCD) had no obvious change. In conclusion, SREBP-1 can activate the expression of genes related to fatty acid synthesis in mammary epithelial cells of Xinong Saanen dairy goat, demonstrated a regulatory function on the fatty acid metabolism in goat mammary gland.
Adenoviridae ; genetics ; metabolism ; Animals ; Epithelial Cells ; cytology ; metabolism ; Fatty Acids ; metabolism ; Female ; Gene Expression Regulation ; Goats ; genetics ; HEK293 Cells ; Humans ; Lipid Metabolism ; genetics ; Mammary Glands, Animal ; cytology ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism ; Transfection

A simple method of trypsin/collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blot, Electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. Effect of hyperthermia on the cell ultrastructures was also observed. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium. After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce apoptosis of the mammary epithelia.
Animals ; Apoptosis ; Blotting, Western ; Caseins ; metabolism ; Cattle ; Cell Line ; Cell Nucleus ; ultrastructure ; Cells, Cultured ; Chromatin ; ultrastructure ; Epithelial Cells ; cytology ; metabolism ; ultrastructure ; Female ; Flow Cytometry ; Hot Temperature ; Immunohistochemistry ; Mammary Glands, Animal ; cytology ; metabolism ; Microscopy, Electron, Transmission ; Mitochondria ; ultrastructure ; Vimentin ; metabolism

During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation.
Animals ; Breast Neoplasms ; genetics ; pathology ; physiopathology ; Cell Transformation, Neoplastic ; Chromatin ; genetics ; metabolism ; Epigenesis, Genetic ; physiology ; Epithelial Cells ; cytology ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Histone-Lysine N-Methyltransferase ; Humans ; Mammary Glands, Animal ; cytology ; growth & development ; Mammary Glands, Human ; cytology ; growth & development ; Myeloid-Lymphoid Leukemia Protein ; genetics ; physiology ; Polycomb-Group Proteins ; genetics ; physiology ; Receptors, Estrogen ; metabolism

To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human beta-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5' and 3' regulating region of beta-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human beta-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human beta-defensin-3 in the supernatant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.
Animals ; Animals, Genetically Modified ; Caseins ; genetics ; Cattle ; Epithelial Cells ; metabolism ; Genes, erbB-1 ; genetics ; Genetic Vectors ; genetics ; Humans ; Mammary Glands, Animal ; cytology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.
Animals ; Anoxia/*genetics/*metabolism ; Caseins/genetics ; Cell Line ; DNA/genetics/metabolism ; DNA-Binding Proteins/*metabolism ; Deferoxamine/pharmacology ; Epithelial Cells/drug effects/*metabolism ; Gene Expression Regulation ; Mammary Glands, Animal/cytology/*metabolism ; Mice ; Phosphorylation/drug effects ; Phosphotyrosine/metabolism ; Promoter Regions (Genetics)/genetics ; Protein Binding ; Response Elements/*genetics ; Support, Non-U.S. Gov't ; Trans-Activators/*metabolism

Curcumin (from the rhizome of Curcuma longa) is well documented for its medicinal properties in Indian and Chinese systems of medicine where it is widely used for the treatment of several diseases. Epidemiological observations are suggestive that curcumin consumption may reduce the risk of some form of cancers and provide other protective biological effects in humans. These biological properties have been attributed to curcuminoids that have been widely studied for their anti-inflammatory, anti-angiogenic, antioxidant, wound healing and anti-cancer effects. In this study we have investigated on the effect of a curcumin phospholipid complex on mammary epithelial cell viability. HC11 and BME-UV cell lines, validated models to study biology of normal, not tumoral, mammary epithelial cells, were used to analyse these effects. We report that curcumin acts on STAT-3 signal pathway to reduce cell viability and increase apoptosis evaluated by the the amount of activated caspase 3. Further it reduces MAPK and AKT activations. JSI-124, a STAT-3 inhibitor (100 nM) was able to block the negative effect of curcumin on cell viability and caspase 3 activation. Finally the negative effect of cucumin on cell viability has been impaired in STAT-3i HC11, where STAT-3 protein was greatly reduced by shRNA-interference. These results indicate that curcumin presents a potential adverse effect to normal mammary epithelial cells and that it has a specific effect on signal trasduction in mammary epithelium.
Animals ; *Apoptosis ; Caspase 3/metabolism ; Cattle ; Cell Differentiation/drug effects ; Cell Survival/drug effects ; Curcuma/chemistry ; Curcumin/*adverse effects ; Enzyme Activation ; Epithelial Cells/cytology/*drug effects ; MAP Kinase Signaling System/physiology ; Mammary Glands, Animal/cytology ; Mice ; Oncogene Protein v-akt/metabolism ; Phospholipids/*pharmacology ; STAT3 Transcription Factor/antagonists & inhibitors/*physiology ; Signal Transduction/drug effects/*physiology ; Triterpenes/pharmacology

The aim of this study was to construct a mammary gland-specific expressional vector pBC1-hLF-Neo for Human Lactoferrin (hLF) gene and then investigate its expression in the mammary gland epithelium cells. The constructed vector contained the 6.2 kb long 5' flank regulation region including promoter, other elements and the 7.1 kb long 3' flank regulation region including transcriptional ending signal of a goat's beta-casein gene. A cassette of Neo gene was also inserted into the vector which gave a total length of 26.736 kb identified by restriction fragment analysis and partial DNA sequencing. The results revealed that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, we transfected the lined vector DNA into the dairy goat's mammary gland epithelium cells and C127 cells of a mouse's mammary epithelium by Lipofectamine. After selection with G418 for 8-10 days, G418-risistant clones were obtained. PCR analysis demonstrated that hLF gene cassette had been integrated into the genomic DNA of G418-risistant clones. After proliferation culture, the two kinds of transgenic cells were cultured in serum-free DMEM-F12 medium with prolactin, insulin and hydrocortisone- a medium capable of inducing recombinant hLF expression. RT-PCR, Western blotting and anti-bacteria bioactivity experiments demonstrated that the constructed mammary gland specific vector pBC1-hLF-Neo possessed the desirable bioactivity to efficiently express and could secrete hLF in both mammary gland cells and have the effect of E. coli proliferation inhibition. Paramount to everything, this study laid a firm foundation for preparing the hLF gene transgenic goat fetal-derived fibroblast cells.
Animals ; Base Sequence ; Breast Neoplasms ; metabolism ; pathology ; Caseins ; genetics ; Cell Line, Tumor ; DNA, Complementary ; biosynthesis ; genetics ; Epithelial Cells ; metabolism ; Female ; Genetic Vectors ; genetics ; Goats ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Mammary Glands, Animal ; cytology ; metabolism ; Mice ; Molecular Sequence Data ; Mutagenesis, Insertional ; Promoter Regions, Genetic ; genetics

Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P < 0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.
Adenoviridae ; genetics ; metabolism ; Animals ; Epithelial Cells ; metabolism ; Female ; Genetic Vectors ; genetics ; Goats ; HEK293 Cells ; Humans ; Mammary Glands, Animal ; cytology ; Parathyroid Hormone-Related Protein ; biosynthesis ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.
Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Caseins ; genetics ; Cloning, Organism ; DNA, Complementary ; genetics ; Gene Knock-In Techniques ; Genetic Engineering ; methods ; Genetic Vectors ; chemical synthesis ; Goats ; genetics ; Humans ; Mammary Glands, Animal ; cytology ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Tissue Plasminogen Activator ; genetics