Abstract
A non-viral gene therapy vector, pcDNA3-EPO, was constructed by subcloning erythropoietin (EPO) cDNA into plasmid pcDNA3. After liposome-mediated transfection of the NIH 3T3 cells in vitro, EPO expression in the culture medium was detected by ELISA and amounted to 1.25 ± 0.3 IU ml−1. The biological activity of this EPO in the medium was detected after intramuscular injection of BALB/c mice. PCR of genomic DNA and RT-PCR of total RNA also confirmed that the plasmid pcDNA3-EPO had been transfected into the cells. A pool of pcDNA3-EPO transfectants, which stably expressed EPO, was obtained by G418 selection. When pcDNA3-EPO was combined into liposomes and intramuscularly injected into BALB/c mice, the reticulocyte ratio in the positive mice was three times higher than that in the control mice. In vivo expression was maintained in mice for at least one month.
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References
Anderson WF (1998) Human gene therapy. Nature 392: 25-30.
Barnhart KM, Hartikka J, Manthorpe M, Norman J, Hobart P (1998) Enhancer and promoter chimeras in plasmids designed for intramuscular injection: a comparative in vivo and in vitro study. Human Gene Ther. 9: 2545-2553.
Campeau P, Chapdelaine P, Seigneurin-Venin S, Massie B, Tremblay JP (2001) Transfection of large plasmids in human myoblasts. Gene Ther. 8: 1387-1394.
Hafez IM, Maurer N, Cullis PR (2001) On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther. 8: 1188-1196.
Hayakawa T, Wada M, Mizuno K, Abe S, Miyashita M, Ueda M (1992) Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins. Biologicals 20: 243-251.
Henry DH, Spivak JL (1995) Clinical use of erythropoietin. Curr. Opin. Hematol. 2: 118-124.
Higuchi M, Oh-eda M, Kuboniwa H, Tomonoh K, Shimonaka Y, Ochi N (1992) Role of sugar chains in the expression of the biological activity of human erythropoietin. J. Biol. Chem. 267: 7703-7709.
Kawasaki ES, Wang AM (1989) Detection of gene expression. In: Erlich HA, ed. PCR Technology, Principles and Applications for DNA Amplification. New York: Stockton Press, pp. 89-97.
Kim JJ, Weiner DB (1999) Development of multicomponent DNA vaccination strategies against HIV. Curr. Opin. Mol. Ther. 1: 43-49.
Kreiss P, Cameron B, Rangara R, Mailhe P, Aguerre-Charriol O, Airiau M, Scherman D, Crouzet J, Pitard B (1999) Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucl. Acids Res. 27: 3792-3798.
Mountain A (2000) Gene therapy: the first decade. Trends Biotechnol. 18: 119-128.
Nagatani N, Shinkai M, Nagase Y, Honda H, Hata K, Mizuno H, Ueda M, Kobayashi T (2000) Gene delivery for genetically engineered mucosal cells with enhanced function. Biotechnol. Lett. 22: 999-1002.
Son KK, Tkach D, Hall KJ (2000) Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA. Biochim. Biophys. Acta 1468: 6-10.
Sytkowski AJ (1984) Erythropoietin: a prime regulator of red cell differentiation. Biomed. Pharm. 38: 369-371.
Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, Kobata A (1988) Comparative study of the asparagine-linked sugar chains of human erythropoietin purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J. Biol. Chem. 263: 3657-3663.
Vile RG, Russell SJ, Lemoine NR (2000) Cancer gene therapy: hard lessons and new courses. Gene Ther. 7: 2-8.
Wang Z, Le G, Shi Y, W¸egrzyn G (2002) Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes. Biotechnol. Lett. 24: 121-124.
Williams DA, Smith FO (2000) Progress in the use of gene transfer methods to treat genetic blood diseases. Human Gene Ther. 11: 2059-2066.
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Zhu, X., Chu, Z., Hu, Y. et al. In vitro and in vivo transfection and expression of plasmid-based non-viral vector for erythropoietin gene therapy. Biotechnology Letters 24, 943–947 (2002). https://doi.org/10.1023/A:1015568931499
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DOI: https://doi.org/10.1023/A:1015568931499