Meili Wang

The interferon-alpha genes from three chicken lines and its effects on H9N2 influenza viruses.

Abstract The interferon-α genes from three chicken lines were cloned by a direct PCR technique, and the effects of recombinant protein expressed in a prokaryotic system on highly pathogenic H9N2 influenza viruses were investigated. The cloned ChIFN-α gene encoded a protein of 193 amino acids with a signal sequence of 31 amino acids and mature peptides of 162 amino acids. Comparison of ChIFN-α sequences, detected six amino acids substitutions at positions 50, 58, 65, 81, 181, and 183. Homology analysis indicated that ChIFN-α genes could be subdivided into two lineages, SH-ChIFN-α and WJ-ChIFN-α. In addition, both SH-ChIFN-α and WJ-ChIFN-α were expressed with the N-terminal 6 consecutive histidine residues in a high-level prokaryotic expression system. Recombinant chicken interferon-α (rChIFN-α) protein has anti-VSV activity of more than 1 × 108 U/mg. Moreover, High concentration (10,000 U) of rSH-ChIFN-α resulted in over 40% inhibition of the H9N2 virus infection in chicken embryos (Ovo), and 100% inhibition from one day-old to five day-old chickens (Vivo). The results suggested that rChIFN-α is a potential agent against many Chicken viral strains.

Maternally transmitted milk containing recombinant human catalase provides protection against oxidation for mouse offspring during lactation.

Catalase plays an important role in protecting organisms against oxidative damage caused by reactive oxygen species (ROS) by degrading surplus hydrogen peroxide. Addition of exogenous catalase can alleviate injuries caused by ROS. Thus, production of human catalase through genetic engineering will meet the increasing therapeutic demand for this enzyme. In this study, we successfully expressed the recombinant gene in mouse mammary gland, and biologically active human catalase was secreted into the milk of the transgenic mice. The peroxisomal targeting sequence (PTS) within the catalase gene had no significant negative effect on the secretion of the recombinant protein. Intake of the transgenic milk by the pups was found to decrease lipid peroxidation, increase the total superoxide dismutase (T-SOD) activity in the brain, and enhance the total antioxidative capacity (T-AOC) of brain, liver, and serum. To our knowledge, this is the first example of efficient production of biologically active human catalase in the milk of transgenic animals. Our study suggests that scaled-up production in transgenic farm animals would yield sufficient human catalase for biomedical research and clinical therapies.

Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups.

Background The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation. Principal Findings Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk. Conclusion Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.

Maternally Derived Recombinant Human Anti-Hantavirus Monoclonal Antibodies Are Transferred to Mouse Offspring during Lactation and Neutralize Virus In Vitro

ABSTRACT Transgenic mice expressing a recombinant human monoclonal antibody (rHMAb) against hantavirus were generated. These mice could be used as models to explore the possibilities of producing rHMAbs for therapeutic purposes. The highest concentration of the rHMAb in the milk of the transgenic females was 6.6 mg/ml. The rHMAb was also detected in the sera of pups fed by the transgenic females. Both the rHMAbs in the milk of transgenic mice and those in the sera of suckling pups were found to be active against hantaviruses, although the light chain of the antibody absorbed by the pups was modified by N-linked glycosylation.

Differential Glycosylation of rhLf Expressed in the Mammary Gland of Transgenic Mice

Differential glycosylation of natural hLf and rhLf from hLf-transgenic mice, which harbored a 146 Kb BAC insert that includes the intact hLf gene sequence, was studied in the present report. There were significant differences between the immunoblotting results of rhLf and natural hLf, which were denatured with nonreducing SDS sample buffer. The differences disappeared after rhLf and natural hLf samples were digested with N-glycosidase F, respectively. The results showed that there were significant differences (P < 0.01) between the glycosylation of natural hLf and rhLf that were purified, respectively, from milk samples of seven hLf-transgenic mouse lines.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D.

Mutation of mevalonate kinase (MVK) is thought to account for most cases of hyperimmunoglobulinemia D syndrome (HIDS) with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D (IgD) and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD (csIgD) specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgenes did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice.

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.

Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

In this study, we introduced the bovine immunoglobulin μ heavy-chain gene (the orphaned gene on BTA11) into mouse germline cells. Bovine IgM was highly expressed in selected transgenic lines, and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain (IgH) genes in these lines. The forced expression of bovine IgM resulted in reduced numbers of proand pre-B cells but increased the number of immature B cells in the transgenic mice. Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen, but most of the cells are arrested at the T1 transitional B cell stage, leading to a significantly lower number of T2 transitional and mature B cells in the spleen. Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased, the IgA levels were slightly increased compared to the WT mice. The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM, suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells. These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3 (CDR3) sequences in their VH repertoire and Vκ repertoire but exhibited an increased frequency of unique CDR3 in their Vλ repertoire. Compared to the WT mice, the transgenic mice had a significantly higher percentage of mouse IgMexpressing B cells that expressed λ chains. Finally, we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.