• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.159-166
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• 2008

We report here the generation of transgenic chickens that produce human Thrombopoietin (hTPO) using replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). For the retrovirus vectors, we used hCMV (human Cytomegalovirus) internal promoter to drive the hTPO gene. After confirming the expression of the hTPO gene in various target cells, the concentrated solution of recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). The biological activity of the recombinant hTPO in target cell was significantly higher than its commercially available counterpart. Out of 132 injected eggs, 11 chicks hatched after 21 days of incubation and 4 hatched chicks were found to express vector-encoded hTPO gene. However, 3 out of the 4 transgenics died within one month of hatching. The major significance of this study is that it is one of the very few successful reports on the production of transgenic chickens as bioreactors aiming mass production of commercially valuable and biological active human cytokine proteins.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.157-162
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• 2006

Endogenous 84 amino acid parathyroid hormone(PTH) is synthesized as a pre-pro hormone by the chief cells of the parathyroid glands. Physiological actions of PTH include regulation of bone metabolism, renal tubular reabsorption of calcium and phosphate, and intestinal calcium absorption. In addition, PTH stimulates new bone formation by extraordinary stimulation of osteoblastic activity and decreasing calcium excretion by the kidney. In this study, we constructed and tested retrovirus vectors designed to express the human parathyroid hormone(hPTH) gene under the control of the tetracycline-inducible promoters. To increase the hPTH gene expression at turn-on state, woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) sequence was also introduced into retrovirus vector at downstream region of either the hPTH gene or the sequence encoding reverse tetracycline-controlled transactivator(rtTA). Transformed primary culture cells(porcine fetal fibroblast, PFF, chicken embryonic fibroblast, CEF) were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the hPTH gene expression level using two step RT-PCR and ELISA Higher hPTH expression($3{\tims}10^4\;pg/ml,\;5.3{\times}10^4\;pg/ml$) and tighter expression control(up to 8 fold) were observed from the vector in which the WPRE sequence was placed at downstream of the hPTH gene. The resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2002.11a
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• pp.139-139
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• 2002

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2001.10b
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• pp.200-200
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• 2001

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.24.1-24.1
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• 2007

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• Korean Journal of Agricultural Science
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• v.36 no.2
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• pp.159-165
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• 2009

This study was conducted to identify the PERV (Porcine Endogenous Retrovirus) integration sites and their characterizations using the porcine genomic sequence information. Total 114 Mb (4.2%) sequence of the 2.7 Gb pig genome was investigated for the PERV sequences. As the results, 8 PERV sequences were identified and their genomic structures were deduced from the BLAST searches against previously known PERV genes. Seven PERVs have internal deletions in the protein coding region and they will not be functional. The other one also has internal deletions in the gag and env genes, indicating this PERV is also defective. Even though we could not identify the functional PERVs in this study, the results presented here can be used for the fundamental research materials for controlling PERV infections in relation to xenotransplantation using porcine organs and tissues.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.91-96
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• 2013

Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time, and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviduct specific expression of human growth hormone fused to human transferrin for oral administration. Gene of the modified growth hormone located at downstream ovalbumin promoter (~3.6 kb) was introduced to stage X blastodermal cell employing retrovirus vector system. Several transgenic chickens were successfully generated. However, genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed to occur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founder animal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, same pattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

• Molecules and Cells
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• v.25 no.1
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• pp.142-147
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• 2008

We recently used degenerate PCR and locus-specific PCR methods to identify the endogenous retroviruses (ERV) in the bovine genome. Using the ovine ERV classification system, the bovine ERVs (BERVs) could be classified into four families. Here, we searched the most recently released bovine genome database with the partial nucleotide sequence of the pro/pol region of the BERV ${\beta}3$ family. This allowed us to obtain and analyze the complete genome of BERV ${\beta}3$. The BERV ${\beta}3$ genome is 7666 nucleotides long and has the typical retroviral organization, namely, 5'-long terminal repeat (LTR)-gag-pro-pol-env-LTR-3'. The deduced open reading frames for gag, pro, pol and env of BERV ${\beta}3$ en- code 507, 271, 879 and 603 amino acids, respectively. BERV ${\beta}3$ showed little amino acid similarity to other betaretroviruses. Phylogenetic analysis showed that it clusters with HERV-K. This is the first report describing the genetic structure and sequence of an entire BERV.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.193-199
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• 2010

We report here the production of transgenic chickens that can regulate human erythropoietin (hEPO) gene expression. The glycoprotein hormone hEPO is an essential for viability and growth of the erythrocytic progenitors. Retrovirus vector system used in this study has two features including tetracycline-controllable promoter and woodchuck hepatitis virus posttranscriptional regulator element (WPRE). The former is for to reduce the possibility of physiological disturbance due to constitutional and unregulated expression of hEPO gene in the transgenic chicken. The latter is for maximum expression of the foreign gene when we turn-on the gene expression. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 325 injected eggs, 28 chicks hatched after 21 days of incubation and 16 hatched chicks were found to express the hEPO gene delivered by the vector. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. The recombinant hEPO in transgenic chicken serum had N- and O-linked carbohydrate simillar to that produced from in vitro cultured cells transformed with hEPO gene.