Xiaojie Wu

Malignant transformation of 293 cells induced by ectopic expression of human Nanog

Tumor development has long been known to resemble abnormal embryogenesis. The ESC self-renewal gene NANOG is purportedly expressed in some epithelial cancer cells and solid tumors, but a casual role in tumor development has remained unclear. In order to more comprehensively elucidate the relationship between human Nanog and tumorigenesis, the hNanog was ectopically expressed in the 293 cell line to investigate its potential for malignant transformation of cells both in vitro and in vivo. Here we provide compelling evidence that the overexpression of hNanog resulted in increased cell proliferation, anchor-independent growth in soft agar, and formation of tumors after subcutaneous injection of athymic nude mice. Pathologic analysis revealed that these tumors were poorly differentiated. In analysis of the underlying molecular mechanism, two proteins, FAK and Ezrin, were identified to be upregulated in the hNanog expressing 293 cells. Our results demonstrate that hNanog is a potent human oncogene and has the ability to induce cellular transformation of human cells.

A mWAP–hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP–hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8xa0g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins.

The high-level accumulation of n-3 polyunsaturated fatty acids in transgenic pigs harboring the n-3 fatty acid desaturase gene from Caenorhabditis briggsae

Livestock meat is generally low in n-3 polyunsaturated fatty acids (PUFAs), which are beneficial to human health. An alternative approach to increasing the levels of n-3 PUFAs in meat is to generate transgenic livestock animals. In this study, we describe the generation of cloned pigs that express the cbr-fat-1 gene from Caenorhabditis briggsae, encoding an n-3 fatty acid desaturase. Analysis of fatty acids demonstrated that the cbr-fat-1 transgenic pigs produced high levels of n-3 fatty acids from n-6 analogs; consequently, a significantly reduced ratio of n-6/n-3 fatty acids was observed. We demonstrated that the n-3 desaturase gene from C. briggsae was functionally expressed, and had a significant effect on the fatty acid composition of the transgenic pigs, which may allow the production of pork enriched in n-3 PUFAs.

The High-Level Expression of Human Tissue Plasminogen Activator in the Milk of Transgenic Mice with Hybrid Gene Locus Strategy

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8xa0g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4xa0kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3xa0g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.

Characterization and Potential Function of a Novel Pre-Implantation Embryo-Specific RING Finger Protein: TRIML1

Members of the super‐class of zinc finger proteins are key regulators in early embryogenesis. Utilizing in silico mining of EST Databases for pre‐implantation Embryo‐Specific Zinc Finger Protein Genes, we characterized a novel zygotic mouse gene—tripartite motif family‐like 1 (TRIML1), which expresses in embryo before implantation. Knocking down of TRIML1 resulted in the fewer cell number of blastocysts and failture to give rise to neonates after embryo transfer. The binding partner of TRIML1, Ubiquitin‐specific protease 5 (USP5), was identified by yeast two‐hybrid screening assay. The interaction was confirmed by GST pull‐down and coimmunoprecipitation analysis. The role of TRIML1 in ubiquitin pathway during the development stage of mouse blastocyst was further discussed. Mol. Reprod. Dev. 76: 656–664, 2009.

The extremely high level expression of human serum albumin in the milk of transgenic mice

The recombinant production of human serum albumin has been challenging due to the low unit price and huge amount needed, for the commercial production of rhSA at an economically feasible level, It will be well worth the effort to exploit new method for the extremely high level expression of rhSA. To this end, here a hybrid gene locus strategy was employed, a 37xa0Kb mWAP-hSA hybrid gene locus was constructed and used as mammary gland specific expression vector, in which the 3xa0Kb genomic coding sequence in the 24xa0Kb mouse whey acidic protein (mWAP) gene locus was substituted by the 16xa0Kb genomic coding sequence of human serum albumin (hSA), exactly from the start codon to the end codon. Corresponding transgenic mice were generated and rhSA was secreted into the milk at an extremely high level of 11.9xa0g/L. Our transgenic mice carrying the mWAP-hSA hybrid gene locus represent a model system for the cost-effective production of human serum albumin.

Plk1-mediated phosphorylation of UAP56 regulates the stability of UAP56

Polo-like kinase 1 (Plk1) is a conserved serine/threonine protein kinase that plays pivotal roles during the cell cycle and cell proliferation. Although a number of important targets have been identified, the mechanism of Plk1-regulated pathways and the bulk of the Plk1 interactome are largely unknown. Here, we demonstrate that Plk1 interacts with the DExH/D RNA helicase, UAP56. The protein levels of UAP56 and Plk1 are inversely correlated during the cell cycle. We also show that Plk1 phosphorylates UAP56 in vitro and in vivo and that Plk1-dependent phosphorylation of UAP56 triggers ubiquitination and degradation of UAP56 through proteasomes. This result suggests that Plk1-mediated phosphorylation of UAP56 regulates the stability of UAP56. Our results will be helpful in further understanding mRNA metabolism, cell cycle progression, and the link between mRNA metabolism and cellular function.

The development of transgenic mice for the expression of large amounts of human lysozyme in milk

Human lysozyme (hLYZ) has important potential applications as antimicrobial medicine and food additive. To develop a robust expression vector that ensures expression of large amounts of hLYZ in milk, here a 26,267xa0bp chimeric mouse whey acidic protein (mWAP)::hLYZ cassette was constructed and used as a mammary gland-specific expression vector, in which a 3,010xa0bp genomic sequence in the 24,466xa0bp mWAP gene locus was substituted by a 4,811xa0bp genomic sequence of hLYZ, exactly from the start codon to the stop codon. Corresponding transgenic mice were generated, and enzymatically-active hLYZ was expressed at 18.4–35xa0gxa0l−1 in the milk of most transgenic mouse lines. Our transgenic mice carrying chimeric mWAP::hLYZ represent a model system for cost-effective production of hLYZ.

NANOG upregulates c-jun oncogene expression through binding the c-jun promoter

NANOG plays important roles in neoplastic processes. However, the molecular mechanism of NANOG in tumorigenesis remains to be elucidated. In this report, we demonstrated that forced expression of NANOG in 293 cells and cancer cells led to increased c‐Jun expression, whereas downregulation of endogenous NANOG significantly reduced c‐Jun expression in cancer cells. Dual luciferase reporter assays demonstrated that NANOG binds the c‐Jun proximal promoter and transactivates the c‐Jun gene. An ATTA consensus motif between the −160 and −268 region of the c‐Jun promoter was identified as the NANOG‐responsive element. Electromobility shift assay and chromatin immunoprecipitation results confirmed the direct binding of NANOG protein to the c‐Jun promoter in vitro and in vivo. NANOG directly bound c‐Jun protein as shown by GST pulldown and immunoprecipitation assays. Taking these findings together, we conclude that c‐Jun is a direct target gene of NANOG and that c‐Jun protein may be a novel co‐activator of NANOG in cancer cells. We suggest the possibility that NANOG may play a significant role in carcinogenesis via its activation of c‐Jun expression.