Avian influenza virus transmission is suppressed in chickens fed Lactobacillus paracasei expressing the 3D8 single-chain variable fragment protein.

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 109 CFU Lactobacillus paracasei (L. paracasei) expressing either a secreted or anchored 3D8 scFv for three weeks. After L. paracasei administration, the chickens were challenged with avian influenza virus (AIV). From each experimental group, three chickens were directly infected with 100 µL of 107.5 EID50/mL H9N2 AIV and seven chickens were indirectly challenged through contact transmission. oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days post-inoculation (dpi) from AIV-challenged chickens, AIV Shedding titres were measured by quantitative real-time PCR. Contact transmission in the chickens that were fed 3D8 scFv-secreting L. paracasei showed a significant reduction in viral shedding when compared with other groups. These results suggest that L. paracasei secreting 3D8 provides a basis for the development of ingestible antiviral probiotics with activity against AIV.

Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission.

The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken ß-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

Transcriptional regulation of cathelicidin genes in chicken bone marrow cells.

Cathelicidins form a family of vertebrate-specific immune molecules with an evolutionarily conserved gene structure. We analyzed the expression patterns of cathelicidin genes (CAMP, CATH3, and CATHB1) in chicken bone marrow cells (BMCs) and chicken embryonic fibroblasts (CEFs). We found that CAMP and CATHB1 were significantly up-regulated in BMCs, whereas the expression of CATH3 did not differ significantly between BMCs and CEFs. To study the mechanism underlying the up-regulation of cathelicidin genes in BMCs, we predicted the transcription factors (TFs) that bind to the 5'-flanking regions of cathelicidin genes. CEBPA, EBF1, HES1, MSX1, and ZIC3 were up-regulated in BMCs compared to CEFs. Subsequently, when a siRNA-mediated knockdown assay was performed for MSX1, the expression of CAMP and CATHB1 was decreased in BMCs. We also showed that the transcriptional activity of the CAMP promoter was decreased by mutation of the MSX1-binding sites present within the 5'-flanking region of CAMP. These results increase our understanding of the regulatory mechanisms controlling cathelicidin genes in BMCs.

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.

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.

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.

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.