Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

Natural and artificially induced mutants have provided valuable resources for plant genetic studies and crop improvement. Some variations induced in the process of plant transformation have often been observed in regenerated plants. In this study, we investigated the insertion number of transgene and the flanking sequences of T-DNA in tall-induced line BP23, which was unexpectedly gained in the process of transformation of insect-resistant rice with cryBP1 gene, and also analyzed the whole-genome sequencing by using the NGS technologies to gain a better understanding of the sequence and structural changes between tall line or natural cultivar and rice reference. than others, was confirmed with two copies of foreign gene insertion, which was inserted in one genomic site facing each other between the position 2,430,152~2,430,151 of rice chromosome 12 without any deletion of genomic sequences. Sequencing analysis also revealed that 18bp-unknown sequences were added in the 5′ insertion site of T-DNA. This position in rice genome was confirmed with none of expressed gene sites. By the NGS analysis, we detected 86560 SNPs and 1091/1472 large insertion/deletion (indel) sites (100bp) between BP23 and rice reference, and 84743 SNPs and 1094/1451 large indels between natural cultivar Nagdong and rice reference. The possible mechanisms for the gene mutation, the developmental and tissue expression of the taller height in BP23 line may need to be scrutinized a few more.

Transgenic plants that over express virus coat protein genes have attracted particular interest from researchers, by virtue of their tolerance to virus infection. The transgenic watermelon rootstock analyzed in this study was established by introducing CGMMV coat protein (cp) under the control of CaMV 35S promoter and NOS terminator (Park et al., (2005) Plant Cell Rep. 24: 350-6). The primary objective of this study was to determine the copy number and integration site of the transgene element, in order to develop detection techniques required for monitoring of the transgenic watermelon rootstock. The Southern blot analysis indicated that a single copy of CGMMV-cp gene was inserted into the genome of transgenic watermelon rootstock. We also identified the genomic sequences flanking the integration site of the transgene by inverse PCR analysis. In an effort to find a sequence usable as an internal positive control for the screening of the watermelon and watermelon rootstock, we found that the Sat and DIP-1 genes appears as one copy within their genomes and is watermelon rootstock- and watermelon-specific. The information of the integrated site and the internal positive control sequence was used to establish a new event-specific PCR-based detection method. In addition, mRNA and protein expression level of the transgene in the transgenic watermelon rootstock and grafted watermelon were investigated. The expression of both mRNA and protein of CGMMV-CP was not detected in the transgenic watermelon rootstocks and watermelons, suggesting that the movement of transgene products from transgenic rootstock to watermelon does not occur at our detection level.