ARTICULOS ORIGINALES

 

Transferencia de genes in vitro con polímeros catiónicos.

 

In vitro gene transfer using cationic polymers

 

Frank Camacho1, Miriela Capo1, Roberto Toledo2, Joel de León3, Arturo Talavera1, Ela M Pérez1.

1Instituto Finlay, Centro de Investigación-Producción de Vacunas. Ave.27 No. 19805. La Lisa. A.P. 16017, C.P. 11600. Ciudad de La Habana, Cuba. E-mail: [e-mail]
2 Centro de Ingeniería Genética y Biotecnología (CIGB). Calle 186 esq. 31, Cubanacán, Playa. A.P. 6162. Ciudad de La Habana, Cuba.
3 Centro de Inmunología Molecular (CIM). Calle 215 esq. 216, Siboney, Playa. Ciudad de La Habana, Cuba.

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RESUMEN

La transferencia de genes representa una importante herramienta para el estudio, prevención y tratamiento de enfermedades genéticas y adquiridas así como para estudios relacionados con la función de proteínas de interés. El uso de moléculas catiónicas está ampliamente difundido, ya que su eficiencia en la transfección las convierte en un fuerte rival de otros métodos de transferencia de genes. En este trabajo se transfectaron las líneas celulares COS-7 y BHK-21 con el plásmido psnEGFP que usó los polímeros catiónicos polietilenimina (PEI) y DEAE dextrana. Al medir la eficiencia de transfección se observó que la línea celular BHK-21 deviene en una excelente opción para la transfección con el método de la PEI y que los mejores resultados se obtienen al disolver PEI en NaCl 150 mM. Al centrifugar las placas después de añadidos los complejos ADN-PEI se obtiene cerca del 100% de células transfectadas con bajas concentraciones de ADN.

Palabras claves: Transfección, polietilenimina, PEI, transferencia de genes, polímeros catiónicos.

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ABSTRACT

Gene transfer represents an important tool for the study, prevention and treatment of genetic and acquired diseases, as well as for protein function studies. Because of their efficiency, the use of cationic molecules has become a strong competitor to other gene transfer methods. In this study, COS-7 and BHK-21 cell lines were transfected with the plasmid psnEGFP using the cationic polymers polyethylene imine (PEI) and DEAE-dextran. When transfection efficiency was measured, it was observed that the cell line BHK-21 is an excellent option for transfection with the PEI method. The best results were obtained when PEI was dissolved in NaCl 150 mM. When plates were centrifuged after addition of the PEI-DNA complexes, nearly 100% of transfected cells with low DNA concentrations were obtained.

Keywords: Transfection, polyethylene imine, PEI, gene transfer, cationic polymers

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REFERENCIAS

1. Motsch I, et al. Lamins A and C are differentially dysfunctional in autosomal dominant Emery-Dreifuss muscular dystrophy. Eur J Cell Biol 2005; 84: 765-81.
2. Horl G, Kroisel PM, Wagner E, Tiran B, Petek E, Steyrer E. Compound heterozygosity (G71R/R140H) in the lecithin: cholesterol acyltransferase (LCAT) gene results in an intermediate phenotype between LCAT-deficiency and fish-eye disease. Atherosclerosis. 2005 Oct 7.
3. Ito S. High frequency of Hermansky-Pudlak syndrome type 1 (HPS1) among Japanese albinism patients and functional analysis of HPS1 mutant protein. J Invest Dermatol 2005; 125:715-20.
4. Toledo JR, Sánchez O, Montesino R, Fernández Y, Rodríguez MP y Cremata JA. Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells. Biochem. Biophys Acta 2005; 1726:48-56.
5. Sanchez O, Toledo JR, Rodríguez MP, Castro FO. Adenoviral vector mediates high expression levels of human growth hormone in the milk of mice and goats. J Biotechnol 2004;114:89-9.
6. Hellebrand E, Mautner J, Reisbach G, Nimmerjahn F, Hallek M, Mocikat R, Hammerschmidt W. Epstein-Barr virus vector-mediated gene transfer into human B cells: potential for antitumor vaccination. Gene Ther 2006; 13:150-62.
7. Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E y Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. U.S.A. 1994; 91:4407-4411.
8. Yang Y, Su Q y Wilson JM. Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduce cells in mouse lungs. J Virol 1996; 70: 7209-7212.
9. Zabner J, Fasbende J, Moninger T, Poellinger A, Welsh M. Cellular and Molecular Barriers to gene transfer by cationic lipid. J Biol Cell 1995; 272:18997-19007.
10. Dunlap D, Maggi A, Soria RM, Monaco L. Nanoscopic structure of DNA condensed for gene delivery. Nucleic Acid Res 1997; 25:3095-3101.
11. Labat-Moleur et al. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 1996; 11:1010-7.
12. Myres MP, Stampe P. A point mutation in the maxi-K clone dSlo forms a high affinity site for charybdotoxin. Neuropharmacology. 2000; 39:11-20.
13. Fujino T, Kang M, Minekura H, Susuki H, Yamamoto T. Alternative translation initiations generates Acyl-CoA synthetase 3 isoforms with heterogeneus amino termini. J Biochem 1997; 122:212-216.
14. Castelli Ch, et al. Novel HLA-Cw8 restricted T cells epitopes derived from Tyrosinase related protein 2 and Gp 100 melanoma antigens. J of Immunol 1998; 162:1739-1744.
15. Boussif O, Zanta MA, Behr JP. Optimized galenics improve in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther. 1996; 3:1074-80.
16. Sambrook J, Fritsch D, Maniatis T. Molecular Cloning. A laboratory manual. Second Edition. Cold Spring Harbor Press. 1984.
17. Goula D, Remy JS, Erbacher P, Wasowicz M, Levi G, Abdallah B, Demeneix BA. Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther 1998; 5:712-7.
18. Godbey WT, Wu KK, Mikos AG. Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery. Proc Natl Acad Sci U S A. 1999; 96:5177-81.
19. Godbey WT, Wu KK, Mikos AG. Poly (ethylenimine) and its role in gene delivery. J Control Release. 1999; 60:149-60.
20. Remy-Kristensen A, Clamme JP, Vuilleumier C, Kuhry JG, Mely Y. Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes. Biochim Biophys Acta. 2001; 1514:21-32.
21. Portelles Y. Optimización de la transfección de células epiteliales mamarias murinas. Tesis de diploma. Universidad de la Habana. Facultad de Biología, Ciudad de La Habana. 1995.
22. Bikales M, Overberger, M. Encyclopedia of polymer Science and Engineering. 1985.