Chul Geun Kim — Page 2

PubMedDate not supplied

Genomic cloning of mud loach Misgurnus mizolepis (Cypriniformes, Cobitidae) beta-actin gene and usefulness of its promoter region for fish transgenesis.

As a part of our effort to obtain a strong regulatory element for the construction of an autogenic mud loach transgenic vector, we isolated a genomic clone that contains an open reading frame encoding the beta-actin gene, then examined the transcriptional activity of the upstream sequences, including the first intron, in transiently transfected cell lines. It showed that the upstream region has substantially strong transcriptional activity, and that both the proximal promoter and distal region of intron 1 play a crucial role in the activity. A similar result, based on fish growth, was obtained with the expression vectors containing the growth hormone gene of mud loach. These were driven by the regulatory region of the mud loach beta-actin gene with various mutations, and were directly transferred into the trunk muscle of fish using an electrostimulation-mediated method. Fish weights were monitored over the next 4 weeks. These data suggest that the proximal promoter and the first intron enhancer of the mud loach beta-actin gene are useful for autogenic mud loach transgenesis.

Actins↗

PubMedDate not supplied

Development of artificial chimerical gene regulatory elements specific for cancer gene therapy.

To achieve satisfactory outcome by the expression of therapeutic genes, it is of great importance to obtain efficient and high level of gene expression as well as minimizing inappropriate gene expression in non-target cells. To accomplish this goal for cancer gene therapy, we have evaluated the potential of cancer specific gene expression of functional promoter/enhancer elements in six putative cancer-specific genes (Tcf1alpha, C-Ha-Ras, CyclinE, Cdc25A, HK II, and hTert) using a luciferase reporter assay. Most of the reporter constructs showed higher activity in HepG2 cells than in non-transformed or stem cells, and, in particular, the hTert (E) or Tcf1alpha (T) regulatory element showed significantly higher activity. We have also constructed a series of artificial chimerical regulatory elements by combinatorial linking of E promoter and T enhancer. A dramatic decrease of activity was observed as the copy number of concatenated T/E regulatory elements increased. In contrast, in chimerical constructs containing two or three copies of regulatory elements of T/E, cell type preferential expression profiles were changed. Thus, both pGL3-TE and -TEE showed higher activity specifically in MCF7 breast cancer cells, whereas pGL3-TET showed moderate activity in several cancer cell lines of different origins. Our results demonstrate that although the transcriptional activities of synthetic promoters are weak, some cancer-specific regulatory elements are useful in developing optimized and systemic cancer-specific regulatory regions with potential application in targeted cancer cell therapy.

Amino Acid Motifs↗