Ik-Soo Jeon

Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector

The Moloney murine leukemia virus (MoMLV) ‐based retrovirus vector system has been used most often in gene transfer work, but has been known to cause silencing of the imported gene in transgenic animals. In the present study, using a MoMLV‐based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of enhanced green fluorescent protein (eGFP). The level of eGFP expression was conserved after germline transmission and as much as 100 g of eGFP could be detected per 1 mg of tissue protein. DNA sequencing showed that the transgene had been integrated at chromosome 26 of the G1 and G2 generation transgenic chickens. Owing to the stable integration of the transgene, it is now feasible to produce G3 generation of homozygous eGFP transgenic chickens that will provide 100% transgenic eggs. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors.—Koo, B. C., Kwon, M. S., Choi, B. R., Kim, J‐H., Cho, S‐K., Sohn, S. H., Cho, E. J., Lee, H. T., Chang, W., Jeon, I., Park, J‐K., Park, J. B., Kim, T. Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV‐based retrovirus vector. FASEB J. 20, 2251–2260 (2006)

Oviduct-Specific Enhanced Green Fluorescent Protein Expression in Transgenic Chickens

In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.

Characterization and miRNA-mediated posttranscriptional regulation of vitelline membrane outer layer protein I in the adult chicken oviduct

The laying hen is the best model for oviduct growth and development. The chicken oviduct produces the egg components, including the egg white and eggshell. However, the mechanism of egg component production during oviduct development requires further investigation. Vitelline membrane outer layer protein 1 (VMO-1) is found in the outer layer of the vitelline membrane of avian eggs. Comparison of the chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins via multiple sequence alignment analysis revealed high degrees of homology of 55%, 53%, 48%, and 54%, respectively. Although the avian homologue of VMO-1 is highly expressed in the magnum of the oviduct, little is known about the transcriptional and posttranscriptional regulation of VMO-1 during oviduct development. The results of this study revealed that estrogen induces VMO-1 messenger RNA (mRNA) expression in oviduct cells in vitro. The expression of genes interacting with VMO-1 by RNA interference (RNAi) functional analysis revealed that ovomucin expression was decreased by VMO-1 silencing. In addition, gga-miR-1623, 1552-3p, and 1651-3p influenced VMO-1 expression via its 3′-UTR, suggesting the posttranscriptional regulation of VMO-1 expression in chickens. Collectively, these results suggest that VMO-1 is an estrogen-induced gene that is posttranscriptionally regulated by microRNAs (miRNAs). The present study may contribute to an understanding of egg component production during chicken oviduct development.

Human extracellular superoxide dismutase (EC-SOD) expression in transgenic chicken.

Extracellular superoxide dismutase (EC-SOD) is a metalloprotein and functions as an antioxidant enzyme. In this study, we used lentiviral vectors to generate transgenic chickens that express the human EC-SOD gene. The recombinant lentiviruses were injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Of 158 injected embryos, 16 chicks (G0) hatched and were screened for the hEC-SOD by PCR. Only 1 chick was identified as a transgenic bird containing the transgene in its germline. This founder (G0) bird was mated with wild-type hens to produce transgenic progeny, and 2 transgenic chicks (G1) were produced. In the generated transgenic hens (G2), the hEC-SOD protein was expressed in the egg white and showed antioxidant activity. These results highlight the potential of the chicken for production of biologically active proteins in egg white. [BMB Reports 2013; 46(8): 404-409]

The gga‐let‐7 family post‐transcriptionally regulates TGFBR1 and LIN28B during the differentiation process in early chick development

Early chick embryogenesis is governed by a complex mechanism involving transcriptional and post‐transcriptional regulation, although how post‐transcriptional processes influence the balance between pluripotency and differentiation during early chick development have not been previously investigated. Here, we characterized the microRNA (miRNA) signature associated with differentiation in the chick embryo, and found that as expression of the gga‐let‐7 family increases through early development, expression of their direct targets, TGFBR1 and LIN28B, decreases; indeed, gga‐let‐7a‐5p and gga‐let‐7b miRNAs directly bind to TGFBR1 and LIN28B transcripts. Our data further indicate that TGFBR1 and LIN28B maintain pluripotency by regulating POUV, NANOG, and CRIPTO. Therefore, gga‐let‐7 miRNAs act as post‐transcriptional regulators of differentiation in blastodermal cells by repressing the expression of the TGFBR1 and LIN28B, which intrinsically controls blastodermal cell differentiation in early chick development. Mol. Reprod. Dev. 82: 967–975, 2015.

The miR-302 cluster transcriptionally regulated by POUV, SOX and STAT5B controls the undifferentiated state through the post-transcriptional repression of PBX3 and E2F7 in early chick development

Early chick development is a systematic process governed by the concerted action of multiple mechanisms that regulate transcription and post‐transcriptional processes. Post‐transcriptional microRNA‐mediated regulation, with regard to lineage specification and differentiation in early chick development, requires further investigation. Here, we characterize the transcriptional and post‐transcriptional regulation mechanisms in undifferentiated chick blastodermal cells. Expression of the miR‐302 cluster, POUV, SOX2, and STAT5B decreased in a time‐dependent manner in early chick development. We found that POUV, SOX2, and STAT5B regulate the transcription of the miR‐302 cluster, as its 5′‐flanking region contains binding elements for each transcription factor. Additionally, POUV, SOX2, and STAT5B maintain pluripotency by regulating genes containing the miR‐302 cluster target sequence. For example, microRNAs from the miR‐302 cluster can bind to PBX3 and E2F7 transcripts, thus acting as a post‐transcriptional regulator that maintains the undifferentiated state of blastodermal cells by balancing the expression of genes related to pluripotency and differentiation. Based on these results, we suggest that both transcriptional and post‐transcriptional regulation of the miR302 cluster is critical for intrinsically controlling the undifferentiated state of chick embryonic blastodermal cells. These findings may help our understanding of the cellular and molecular mechanisms that underlie developmental decisions during early chick development. Mol. Reprod. Dev. 81: 1103–1114, 2014.

STAT5 plays a critical role in regulating the 5′‐flanking region of the porcine whey acidic protein gene in transgenic mice

The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical‐grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5′‐flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene‐expression activity. In the present study, we investigated how lactogenic hormones—including prolactin, hydrocortisone, and insulin—contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5′‐flanking region. To confirm the expression and hormonal regulation of the 5′‐flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk. Mol. Reprod. Dev. 82: 957–966, 2015.