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Development and validation of transgene constructs in order to generate bi-transgenic mice models for Diabetic Peripheral Neuropathy research
Nair, Lakshmy
Nair, Lakshmy
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Abstract
Diabetic peripheral neuropathy (DPN), a common complication of diabetes, involves nerve damage in the arms and legs. Segmental demyelination is one of the basic patterns observed in the pathology of DPN. Demyelinating neuropathies are those in which the Schwann cells (SCs) are primarily affected and these undergo substantial degeneration in diabetic neuropathy. Hence, it is of pertinence to investigate possible mechanisms which may contribute to the demyelination of SCs and the progression of DPN. To address this issue, this project aims to generate three different bi-transgenic mouse models that provide for the SC-specific expression of several transgenes that can be induced by the addition of the antibiotic doxycycline. For temporal, spatial and cell-specific control of transgene expression in mice, the above mentioned transgenes were constructed based on the Tet-On gene regulation system. In order to yield spatially regulated transgene expression, rtTA (reverse tetracycline transactivator) was placed under the control of the SC-specific promoter for 2', 3'-cyclic nucleotide 3'-phosphodiesterase. Placement of genes under the control of the rtTA- Ptight system has been shown to display excellent dose-response characteristics, which allows not only a qualitative off-on transition but also a fine tuning of gene expression and the study of quantitative aspects of gene activity. The above mentioned transgenes constructed in this project were validated in Hek293T cells to ensure that they expressed in a tight and highly inducible manner. The transgene transfected cell lines when treated with doxycycline showed a dose-dependent expression of the gene of interest. Maximum gene induction was observed when treated with 0.5ug/ml doxycycline. In the absence of doxycycline, almost no or minimal gene expression was observed. The transgenes were then excised out of the vector backbone for pronuclear microinjection. Once the transgenic mice are born, identified and validated, they shall be used for diabetic peripheral neuropathy research in the laboratory.
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Date
2010-04-09
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University of Kansas
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Keywords
Molecular biology, Biology, Neurosciences, Cell biology, Diabetic peripheral neuropathy, Schwann cells, Tet-on gene regulation system, Transgene construction, Transgene expression, Transgenic mouse model