Abstract
Our lab has constructed a new nonviral vector—hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hFVIII (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII-BDDAK39 in the hrDNA locus. The hFVIII-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 2.0×10−5. hFVIII-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells−1 · 24 h−1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chen, C., Fang, X. D., Zhu, J. et al., The gene expression of coagulation factor VIII in mammalian cell lines, Thrombosis Res., 1999, 95: 105–115.
Plantier, J. L., Rodriguez, M. H., Enjolras, N. et al., A factor VDI minigene comprising the truncated intron I of factor IX highly improves thein vitro production of factor VIII, Thromb. Haemost., 2001, 86(2): 596–603.
Lynch, C. M., Israel, D. I., Kaufman, R. J. et al., Sequences in the coding region of clotting factor VIII act as dominant inhibitors of RNA accumulation and protein production, Hum. Gene Ther., 1993, 4(3): 259–272.
Hoeben, R. C., Fallaux, F. J., Cramer, S. J. et al., Expression of the blood-clotting factor-VIII cDNA is repressed by a transcriptional silencer located in its coding region, Blood, 1995, 85(9): 2447–2454.
Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10(13): 2091–2107.
Kaufman, R. J., Pipe, S. W., Regulation of factor VIII expression and activity by von Willebrand factor, Thromb. Haemost., 1999, 82(2): 201–208.
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989.
Brown, J. P., Wei, W., Sedivy, J. M., Bypass of senescence after disruption of p21 CIP1/WAF1 gene in normal diploid human fibroblasts, Science, 1997, 277(5327): 831–834.
Arbones, M. L., Austin, H. A., Capon, D. J. et al., Gene targeting in normal somatic cells: Inactivation of the interferon-gamma receptor in myoblasts, Nat. Genet., 1994, 6(1): 90–97.
Hanson, K. D., Sedivy, J. M., Analysis of biological selections for high efficiency gene targeting, Mol. Cell Biol., 1995, 15(1): 45–51.
Opes, T. S., Klootwijk, J., Veenstra, A. E. et al., High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: A new vector for high-level expression, Gene, 1989, 79(2): 199–206.
Wery, J., Gutker, D., Renniers, A. C. et al., High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma, Gene, 1997, 184(1): 89–97.
Santos, W. G., Buck, G. A., Simultaneous stable expression of neomycin phosphotransferase and green fluorescence protein genes inTrypanosoma cruzi, J. Parasitol., 2000, 86(6): 1281–1288.
Steele, R. E., Bakken, A. H., Reeder, R. H., Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells, Mol. Cell Biol., 1984, 4(4): 576–582.
Stewart, S., MacDonald, N., Perkins, E. et al., Retrofitting of a satellite repeat DNA-based murine artificial chromosome (ACes) to contain loxP recombination sites, Gene Ther., 2002, 9(11): 719–723.
Sedivy, J. M., Dutriaux, A., Gene targeting and somatic cell genetics—A rebirth or a coming of age? Trends Genet., 1999, 15(3): 88–90.
Szostak, J. W., Wu, R., Unequal crossing over in the ribosomal DNA ofSaccharomyces cerevisiae, Nature, 1980, 284(5755): 426–430.
Brooks, A. R., Harkins, R. N., Wang, P., Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle, J. Gene Med., 2004, 6(4): 395–404.
Richards, B., Zharkikh, L., Hsu, F. et al., Stable expression of Anthozoa fluorescent proteins in mammalian cells, Cytometry, 2002, 48(2): 106–112.
Fleury, S., Simeoni, E., Zuppinger, C., Multiply attenuated, self-inactivating lentiviral vectors efficiently deliver and express genes for extended periods of time in adult rat cardiomyocytesin vivo, Circulation, 2003, 107(18): 2375–2382.
Chen, L., Chen, H. M., Zou, B. Y. et al., Preparation of rAAV/hFIX by HSV/AAV hybrid helper virus and evaluation of its safety, Chinese Science Bulletin, 2003, 48(13): 1369–1374.
Jenke, A. C., Scinteie, M. F., Stehle, I. M., Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation, Mol. Biol. Rep., 2004, 31(2): 85–90.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to this work.
About this article
Cite this article
Liu, X., Liu, M., Hua, S. et al. Expression of reconstructive hFVIII in the hrDNA by using hrDNA targeting vector. Chin.Sci.Bull. 50, 2187–2192 (2005). https://doi.org/10.1007/BF03182670
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF03182670