To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5' flanking sequence, exon Ⅰ, intron Ⅰ an...To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5' flanking sequence, exon Ⅰ, intron Ⅰ and exon Ⅱ. A 661-bp region of the cloned fragment, consisting of the 650-bp 5' flanking sequence and a non-coding sequence of 11 bp downstream of the transcription initiation site, was used as a regulatory element to combine with human growth hormone (hGH) gene to generate a bovine BLG/hGH fusion construct, which was then introduced into cultured primary mammary epithelial cells of goat for transient expression of hGH gene. It was demonstrated that the hGH gene was able to express following hormone induction and the expressed product was able to be secreted into the medium. The bovine BLG/hGH fusion construct was also used to generate transgenic mice by microinjection. Subsequently, five transgenic mice were generated. The hGH in milk by one transgenic female mouse was 420μg/mL, while the content of hGH in serum was 0.051 μg/mL only. This indicated that the cloned regulatory element in this experiment could make the expression of target gene occur almost specifically in mammary gland and the expressed product could be secreted with milk.展开更多
The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often t...The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.展开更多
and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BL...and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BLG 5′ and 3′ flanking were the same as that of publication completely.The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2kb 5′ flanking with 2.1kb 3′ flanking.In order to assess the efficiency of BLG regulatory elements,green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47).Through observation under UV microscope and detection by fluorometer,it is demonstrated that the GFP has been successfully expressed in TD47 cell line.By virtue of direct observation and quantitative analysis,the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.展开更多
文摘To obtain a regulatory element for generating mammary bioreactors. a DNA fragment derived from bovine β-lactoglobulin (BLG) gene was cloned, which consisted of a 650-bp 5' flanking sequence, exon Ⅰ, intron Ⅰ and exon Ⅱ. A 661-bp region of the cloned fragment, consisting of the 650-bp 5' flanking sequence and a non-coding sequence of 11 bp downstream of the transcription initiation site, was used as a regulatory element to combine with human growth hormone (hGH) gene to generate a bovine BLG/hGH fusion construct, which was then introduced into cultured primary mammary epithelial cells of goat for transient expression of hGH gene. It was demonstrated that the hGH gene was able to express following hormone induction and the expressed product was able to be secreted into the medium. The bovine BLG/hGH fusion construct was also used to generate transgenic mice by microinjection. Subsequently, five transgenic mice were generated. The hGH in milk by one transgenic female mouse was 420μg/mL, while the content of hGH in serum was 0.051 μg/mL only. This indicated that the cloned regulatory element in this experiment could make the expression of target gene occur almost specifically in mammary gland and the expressed product could be secreted with milk.
文摘The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.
文摘and 3′ flanking region of ovine BLG were amplified from sheep genomic DNA according to the published whole sequence of ovine BLG and cloned to pGEM-T vector correspondently.By partially sequencing,the sequences of BLG 5′ and 3′ flanking were the same as that of publication completely.The recombinant structure used to direct exogenous gene especially to express in mammary gland was constructed by joining 4.2kb 5′ flanking with 2.1kb 3′ flanking.In order to assess the efficiency of BLG regulatory elements,green fluorescent protein (GFP) gene as a reporter was fused with BLG construct and transfected the mammary epithelial cells (TD47).Through observation under UV microscope and detection by fluorometer,it is demonstrated that the GFP has been successfully expressed in TD47 cell line.By virtue of direct observation and quantitative analysis,the BLG-GFP construct can be served as a model for the quick assessment of mammary gland expression construct.
