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

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

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

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

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.
Animals ; Caseins/metabolism ; Cathepsin B/antagonists&inhibitors/*genetics/isolation & purification/*metabolism ; Cysteine Proteinase Inhibitors/pharmacology ; DNA, Complementary/genetics ; Goat Diseases/*parasitology ; Goats ; Haemonchiasis/parasitology/*veterinary ; Haemonchus/*enzymology/genetics/isolation & purification ; Hemagglutination Tests/veterinary ; Hemoglobins/metabolism ; Hydrogen-Ion Concentration ; Immunoglobulin G/metabolism ; Leucine/analogs & derivatives/pharmacology ; RNA, Helminth/chemistry/genetics ; Recombinant Proteins/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/veterinary

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

This study focused on the contamination mechanism and regeneration strategies of sulfopropyl ion exchange resin (SP Sepharose FF) during the separation of recombinant human lactoferrin from transgenic bovine milk. We analyzed primary constituents' contents in chromatorgraphic material and fractions. The results showed that the lipid in milk can clog the column or adhere to the resin through hydrophobic interaction, leading to an increase in column pressure. Some casein molecules were found to adsorb onto the resin through electrostatic interaction, therefore the adsorption capacity was decreased. There was no direct interaction between lactose and the resin in the chromatorgraphic process. Increased continuous chromatographic cycles and prolonged time interval between protein purification and column regeneration could enhance the undesirable interaction between the contaminants and resin, thus lowering the regeneration efficiency. NaOH was found to be effective in the removal of lipid and casein molecules from the column. Furthermore, normal microstructure and chromatographic performance of the ion exchanger was recovered after this cleaning procedure.
Adsorption ; Animals ; Animals, Genetically Modified ; genetics ; metabolism ; Caseins ; chemistry ; Cattle ; Chromatography, Ion Exchange ; methods ; Female ; Humans ; Ion Exchange Resins ; chemistry ; Lactoferrin ; biosynthesis ; genetics ; isolation & purification ; Mammary Glands, Animal ; metabolism ; Milk ; chemistry ; Milk Proteins ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Hydroxide ; chemistry

OBJECTIVETo establish an appropriate neonatal rat model of necrotizing enterocolitis (NEC) and to investigate the protective effects of glycomacropeptide (GMP) on the gut from injury in neonatal rats with NEC.

METHODA total of 36 neonatal SD rats were randomly divided into 3 groups: NEC model group (Group M), NEC + GMP group (Group G) and normal control group (Group N), each group had 12 rats. All the neonatal rats were fed with breast milk in the first 3 days after birth. During the second 3 days after birth, the rats of Group N were still maternal breast-fed, but the rats of Group M and Group G were separated from their mothers and lived in incubator and began to be formula fed, and were subjected to cold exposure shortly after hypoxic-reoxygenation treatment. After being fed in such means for 6 days, all the neonatal rats were placed into the incubator and fasted for 24 hours. Then all the rats were sacrificed by cervical dislocation. Intestinal tissue located at the boundary of ileum and cecum was obtained for: (1) histological examination after HE staining, (2) TUNEL detection, (3) electron microscopic observation; and the tissue homogenate was obtained for checking TNF-α and IL-1β levels by ELISA and platelet activating factor (PAF) mRNA expression by quantitative fluorescence (QF)-PCR.

RESULT(1) The pathological scores of the 3 groups were 2.17 ± 0.83 (Group M), 0.92 ± 0.79 (Group G) and 0.17 ± 0.39 (Group N) separately. There was significant difference between Group M and Group G (H = 8.819, P = 0.003). (2) TNF-α levels of 3 groups were (41.94 ± 13.51) pg/ml (Group M), (31.69 ± 11.68) pg/ml (Group G) and (17.42 ± 7.18) pg/ml (Group N) separately, and TNF-α level in Group G was significantly lower than that of Group M (F = 3.959, P = 0.030). (3) IL-1β levels of 3 groups were (150.33 ± 36.41) pg/ml (Group M), (118.36 ± 33.00) pg/ml (Group G) and (28.44 ± 15.04) pg/ml (Group N) separately, and IL-1β level in Group G was lower than that of Group M (F = 5.080, P = 0.013). (4) Expression levels of intestinal PAF mRNA (2(-ΔΔCt) value): 3.01 ± 0.96 (Group M), 1.56 ± 0.29 (Group G), 1.01 ± 0.13 (Group N), the level of Group G was significantly lower than that of Group M (F = 25.251, P = 0.000). (5)Electron microscopy: Group N showed that its cell volume was mostly occupied by the nucleus, the structure was clear, nuclear membrane existed, suggesting the normal phase of cell; Group M showed that apoptotic body existed, suggesting that the advanced stage phase of apoptosis; Group G showed that condensed chromatin marginated around the nuclear envelope, nuclear pores expanded, suggesting the early phase of apoptosis. (6) The apoptosis rate of intestinal epithelial cells by TUNEL detection: 38.79 ± 9.79 (Group M), 29.54 ± 7.30 (Group G), 6.37 ± 1.96 (Group N); the apoptosis rate of intestinal epithelial cells of Group G was significantly lower than that of Group M (F = 6.888, P = 0.003).

CONCLUSIONGMP has protective effects on guts of neonatal rats with NEC, which may probably work by reducing TNF-α, IL-1β and PAF expression, inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury.

Animals ; Animals, Newborn ; Apoptosis ; Caseins ; pharmacology ; Cold Temperature ; Enterocolitis, Necrotizing ; drug therapy ; metabolism ; pathology ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Female ; Hypoxia ; complications ; Interleukin-1beta ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; metabolism ; pathology ; Male ; Peptide Fragments ; pharmacology ; Platelet Activating Factor ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism