Abstract
Aim: Future prospects for gene therapy of renal anemia involve expression of the EPO transgene. Control of gene expression is important to gene therapy for purposes of both dosing and safety. We tested the effects of rhEPO gene expression under the tetracycline gene expression system’s regulating and controlling. Methods: Cultured rat primary skeletal muscle cells for gene delivery use; Inserted the hEPO cDNA in the plasmid pTRE2hyg; Rat primary skeletal muscle cells were engineered for Dox-inducible and skeletal muscle-specific expression of rhEPO cDNA by co-transfection pTRE2hyg-EPO and Tet-ON plasmids. Results: EPO could be secreted by the transfected positive cells. EPO expression depended on the presence of doxycycline, and the EPO secretion was increased parallel to dose of doxycycline. Conclusion: Tetracycline gene expression system provided a suitable control system for sustained and regulated expression of a desired gene.
Highlights
Erythropoietin (EPO) is a 30-kd glycoprotein hormone that is the regulator of red blood cell production and maintenance in mammals. [1,2] In human adults, the erythropcyte-generating hormone, is produced mainly by the kidney peritubular cells [3]
EPO expression depended on the presence of doxycycline, and the EPO secretion was increased parallel to dose of doxycycline
Anaemia is a major complication of chronic renal failure (CRF), which results from a destruction of EPO-secreting cells
Summary
Erythropoietin (EPO) is a 30-kd glycoprotein hormone that is the regulator of red blood cell production and maintenance in mammals. [1,2] In human adults, the erythropcyte-generating hormone, is produced mainly by the kidney peritubular cells [3]. Erythropoietin (EPO) is a 30-kd glycoprotein hormone that is the regulator of red blood cell production and maintenance in mammals. The administration of recombinant EPO is widely used for long-term treatment of anemia associated with chronic renal failure, cancer chemotherapy, and HIV infections. [4] this treatment requires repeated administration of recombinant protein, which is both inconvenient and expensive [5]. Delivery of this hormone by gene therapy rather than. To model an essential gene under tetracycline regulation, the hygromycin resistance gene (hph) was placed under the control of seven copies of the TetR binding site (tetO) in a plasmid vector and co-transfected into cells together with one of the two transactivator plasmids. Transformants were identified in which the expression of tTA conferred hygromycin resistance by activating expression of the tetO-hph reporter gene[26]
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