Establishment of canine kidney cell line for canine distemper virus replication / 동물의과학연구지

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

The expression of Foxp3 protein by retroviral vector-mediated gene transfer of Foxp3 in C57BL/6 mice / 대한수의학회지

The maintenance of peripheral immune tolerance and prevention of chronic inflammation and autoimmune disease require CD4+CD25+ T cells (regulatory T cells). The transcription factor Foxp3 is essential for the development of functional, regulatory T cells, which plays a prominent role in self-tolerance. Retroviral vectors can confer high level of gene transfer and transgene expression in a variety of cell types. Here we observed that following retroviral vector-mediated gene transfer of Foxp3, transductional Foxp3 expression was increased in the liver, lung, brain, heart, muscle, spinal cord, kidney and spleen. One day after vector administration, high levels of transgene and gene expression were observed in liver and lung. At 2 days after injection, transductional Foxp3 expression level was increased in brain, heart, muscle and spinal cord, but kidney and spleen exhibited a consistent low level. This finding was inconsistent with the increase in both CD4+CD25+ T cell and CD4+Foxp3+ T cell frequencies observed in peripheral immune cells by fluorescence-activated cell-sorting (FACS) analysis. Retroviral vector-mediated gene transfer of Foxp3 did not lead to increased numbers of CD4+CD25+ T cell and CD4+Foxp3+ T cell. These results demonstrate the level and duration of transductional Foxp3 gene expression in various tissues. A better understanding of Foxp3 regulation can be useful in dissecting the cause of regulatory T cells dysfunction in several autoimmune diseases and raise the possibility of enhancing suppressive functions of regulatory T cells for therapeutic purposes.