• Korean Journal of Poultry Science
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• v.36 no.2
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• pp.177-186
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• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Journal of Life Science
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• v.28 no.4
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• pp.389-397
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• 2018

The structure of glycan residues attached to glycoproteins can influence the biological activity, stability, and safety of pharmaceutical proteins delivered from transgenic pig milk. The production of therapeutic glycoprotein in transgenic livestock animals is limited, as the glycosylation of mammary gland cells and the production of glycoproteins with the desired homogeneous glycoform remain a challenge. The ${\beta}$-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) gene is an important enzyme that attaches N-acetylglucosamine (GlcNAc) to galactose (Gal) residues for protein glycosylation; however, there is limited information about pig glycosyltransferases. Therefore, we cloned the pig B3GNT1 (pB3GNT1) and investigated its functional properties that could attach N-acetylglucosamine to galactose residue. Using several different primers, a partial pB3GNT1 mRNA sequence containing the full open reading frame (ORF) was isolated from liver tissue. The ORF of pB3GNT1 contained 1,248 nucleotides and encoded 415 amino acid residues. Organ-dependent expression of the pB3GNT1 gene was confirmed in various organs from adult and juvenile pigs. The pB3GNT1 mRNA expression level was high in the muscles of the heart and small intestine but was lower in the lungs. For functional characterization of pB3GNT1, we established a stable expression of the pB3GNT1 gene in the porcine kidney cell line (PK-15). As a result, it was suggested that the glycosylation pattern of pB3GNT1 expression in PK-15 cells did not affect the total sialic acid level but increased the poly N-acetyllactosamine level. The results of this study can be used to produce glycoproteins with improved properties and therapeutic potential for the generation of desired glycosylation using transgenic pigs as bioreactors.

• Journal of Korean Society of Environmental Engineers
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• v.27 no.5
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• pp.499-506
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• 2005

This study was performed to investigate the effects of influent C/N ratio on the removal of organic and nitrogenous compounds by two nonwoven fabric filter bioreactors. The reactors were alternately aerated at an aeration/nonaeration period ratio of 60 min/60 min, and fed with wastewater only during nonaeration period. The influent C/N ratio (COD/TKN) was gradually reduced from 10 to 2. The influent was prepared by diluting the leachate from a foodwaste treatment facility in I city so that the COD concentration could be about 2,500 mg/L. The C/N ratio of the wastewater was adjusted by adding ammonium chloride. The results of the experiment showed that the COD and BOD concentration of the effluent was $40{\sim}54\;mg/L$ and $1{\sim}4\;mg/L$, respectively at the C/N ratios of $10{\sim}3$, and the effluent SS concentration was always below 2.0 mg/L. The T-N removal efficiencies were 96% or higher at C/N ratios of $10{\sim}5$, but decreased to 83% and 81%, respectively at the C/N ratios of 3 and 2.8. At the C/N ratios of 2.6 and 2, the effluent quality deteriorated due to ammonia toxicity. The fraction of nitrifying microorganism in the reactors increased from 10% to 20% as the C/N ratio decreased from 5 to 2.6. Alkalinity consumed were $3.12{\sim}3.49\;g$ alkalinity/g T-N removed at the C/N ratios of $10{\sim}5$, which are lower than the theoretical value of 3.57. However, the ratio increased to 4.63 and 4.87 g alkalinity/g T-N removed, respectively at the C/N ratios of 3 and 2.8.