ABSTRACT
A few years ago, three novel murine homeobox genes closely related to the Drosophila sine oculis (so) gene (Six1-3) were isolated and were all included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional ortholog of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue. Here, we show that similar to Drosophila, where the eyeless/Pax6 gene regulates the eye-specific expression of so, Six3 expression in the murine lens placodal ectoderm is also controlled by Pax6. We also show that ectopic Six3 expression promotes the formation of ectopic optic vesicle-like structures in the hindbrain-midbrain region of developing mouse embryos.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Lens, Crystalline/embryology , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , DNA-Binding Proteins/genetics , Ectoderm/metabolism , Embryonic and Fetal Development , Eye Proteins , Head/embryology , Homeodomain Proteins/biosynthesis , Lens, Crystalline/metabolism , Mice , Mice, Mutant Strains , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , PAX2 Transcription Factor , PAX6 Transcription Factor , Paired Box Transcription Factors , Repressor Proteins , Time Factors , Transcription Factors/genetics , Homeobox Protein SIX3ABSTRACT
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.
Subject(s)
Gene Expression Regulation/physiology , Interferon-gamma/genetics , Lactoglobulins/genetics , Mammary Glands, Animal/metabolism , Animals , Female , Humans , Hybridization, Genetic , Mice , Mice, Transgenic , Milk/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.
Subject(s)
Glycopeptides/analysis , Interferon-gamma/metabolism , Oligosaccharides/analysis , Animals , Baculoviridae , CHO Cells , Carbohydrate Conformation , Cell Line , Cricetinae , Female , Glycosylation , Humans , Insecta/cytology , Insecta/virology , Mammary Glands, Animal/metabolism , Mice , Mice, Transgenic , Recombinant Proteins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.
