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

Sung YY, Lee CS - (2013)

Bottom Line: An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer.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.

View Article: PubMed Central - PubMed

Affiliation: Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Construction of hOPG cDNA expression cassette. A, Goat β-casein/hGH hybrid gene, which was originally described by Lee (2006). B, Modified goat β-casein/hGH hybrid gene for cDNA expression. Internal fragment between two PvuII sites in hGH gene was deleted and two ATG codons in hGH exon I and II were mutated to SalI and HpaI cloning sites, respectively. C, hOPG cDNA expression cassette. The amplified hOPG cDNA sequences were inserted into the HpaI cloning site for its mammary gland-specific expression in transgenic mice. The 5’ flanking and promoter sequences of goat β-casein gene is represented as a bold line. Exons of hGH gene are blanked. Poly(A) and 3’ flanking sequences of hGH gene are shaded. hOPG cDNA sequences are filled.
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Figure 1: Construction of hOPG cDNA expression cassette. A, Goat β-casein/hGH hybrid gene, which was originally described by Lee (2006). B, Modified goat β-casein/hGH hybrid gene for cDNA expression. Internal fragment between two PvuII sites in hGH gene was deleted and two ATG codons in hGH exon I and II were mutated to SalI and HpaI cloning sites, respectively. C, hOPG cDNA expression cassette. The amplified hOPG cDNA sequences were inserted into the HpaI cloning site for its mammary gland-specific expression in transgenic mice. The 5’ flanking and promoter sequences of goat β-casein gene is represented as a bold line. Exons of hGH gene are blanked. Poly(A) and 3’ flanking sequences of hGH gene are shaded. hOPG cDNA sequences are filled.

Mentions: For an efficient and mammary gland-specific expression of hOPG from its cDNA sequences, a cDNA expression cassette was designed by modifying the goat β-casein/hGH hybrid gene construct, which previously directed a strong and mammary gland-specific expression of hGH in transgenic mice (Lee, 2006). The goat β-casein/hGH hybrid gene construct consists of 5’ promoter sequence (5.5 kb) of goat β-casein gene and entire structural sequence (2.1 kb) of hGH gene (Fig. 1A). In order to modify this hybrid gene for hOPG cDNA expression, internal pvuII fragment (984 bp) between exon II and exon V of hGH gene was excised out and ATG codons at exon I and II were replaced with SalI and HpaI cloning sites by PCR using a primer set (5'-AGCTGTCGACGCTACAGGTAAG-3' and 5'-GGCCAGCTGGTGTTAACGATGGGCGCGGAGGAT AGCG-3'). This modification generated a novel cDNA expression cassette (Fig. 1B) containing goat β-casein promoter sequences and exon I and II, intron I, poly (A), and 3’ flanking sequences of hGH gene with no ATG start codon. For hOPG expression, full-length hOPG cDNA (1,206 bp) was amplified by PCR using a primer set (5'-TCCCGGGGACCACAATGAAC-3' and 5'-GGTCGAC TTATAAGCAGCTTATTT-3') and inserted into HpaI site of the cassette by blunt ligation (Fig. 1C). Correct nucleotide sequence of this hOPG expression cassette was confirmed by sequencing before use for DNA microinjection.


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

Sung YY, Lee CS - (2013)

Construction of hOPG cDNA expression cassette. A, Goat β-casein/hGH hybrid gene, which was originally described by Lee (2006). B, Modified goat β-casein/hGH hybrid gene for cDNA expression. Internal fragment between two PvuII sites in hGH gene was deleted and two ATG codons in hGH exon I and II were mutated to SalI and HpaI cloning sites, respectively. C, hOPG cDNA expression cassette. The amplified hOPG cDNA sequences were inserted into the HpaI cloning site for its mammary gland-specific expression in transgenic mice. The 5’ flanking and promoter sequences of goat β-casein gene is represented as a bold line. Exons of hGH gene are blanked. Poly(A) and 3’ flanking sequences of hGH gene are shaded. hOPG cDNA sequences are filled.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282218&req=5

Figure 1: Construction of hOPG cDNA expression cassette. A, Goat β-casein/hGH hybrid gene, which was originally described by Lee (2006). B, Modified goat β-casein/hGH hybrid gene for cDNA expression. Internal fragment between two PvuII sites in hGH gene was deleted and two ATG codons in hGH exon I and II were mutated to SalI and HpaI cloning sites, respectively. C, hOPG cDNA expression cassette. The amplified hOPG cDNA sequences were inserted into the HpaI cloning site for its mammary gland-specific expression in transgenic mice. The 5’ flanking and promoter sequences of goat β-casein gene is represented as a bold line. Exons of hGH gene are blanked. Poly(A) and 3’ flanking sequences of hGH gene are shaded. hOPG cDNA sequences are filled.
Mentions: For an efficient and mammary gland-specific expression of hOPG from its cDNA sequences, a cDNA expression cassette was designed by modifying the goat β-casein/hGH hybrid gene construct, which previously directed a strong and mammary gland-specific expression of hGH in transgenic mice (Lee, 2006). The goat β-casein/hGH hybrid gene construct consists of 5’ promoter sequence (5.5 kb) of goat β-casein gene and entire structural sequence (2.1 kb) of hGH gene (Fig. 1A). In order to modify this hybrid gene for hOPG cDNA expression, internal pvuII fragment (984 bp) between exon II and exon V of hGH gene was excised out and ATG codons at exon I and II were replaced with SalI and HpaI cloning sites by PCR using a primer set (5'-AGCTGTCGACGCTACAGGTAAG-3' and 5'-GGCCAGCTGGTGTTAACGATGGGCGCGGAGGAT AGCG-3'). This modification generated a novel cDNA expression cassette (Fig. 1B) containing goat β-casein promoter sequences and exon I and II, intron I, poly (A), and 3’ flanking sequences of hGH gene with no ATG start codon. For hOPG expression, full-length hOPG cDNA (1,206 bp) was amplified by PCR using a primer set (5'-TCCCGGGGACCACAATGAAC-3' and 5'-GGTCGAC TTATAAGCAGCTTATTT-3') and inserted into HpaI site of the cassette by blunt ligation (Fig. 1C). Correct nucleotide sequence of this hOPG expression cassette was confirmed by sequencing before use for DNA microinjection.

Bottom Line: An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer.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.

View Article: PubMed Central - PubMed

Affiliation: Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811, Korea.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus