Construction of Recombinant Adenovirus Vector with Human/Mouse Interferon Gene and Its Transfection intoHuman Umbilical Cord Mesenchymal Stem Cells

LI Hui, HU Qiong-ying, JIA Yong-qian

Abstract

Construction and identification of recombinant adenovirus vector with human/mouse interferon (IFN-γ) and effectively transfection into human umbilical cord mesenchymal stem cells (HUMSCs). Methods IFN-γ gene of human/mouse were amplified from the plasmid by polymerase chain reaction (PCR) and then inserted into the plasmid pDC316 to generate pDC316-IFN-γ. After being confirmed by restriction enzyme digestion and DNA sequencing, the DNA encoding IFN-γ in the new structure were inserted into the vector of recombinant plasmid adenovirus and confirmed by restricition enzyme digestion. Then the human embryonic kidney cell line 293 were transfected with confirmed Ad-IFN-γ, and the recombinant adenovirus were amplified, and the virus titer were detected using 50% tissue culture infective dose (TCID50) assay. The expression of the green fluorescent protein (GFP) and IFN-γ were detected by fluorescent microsope and Western blot and ELISA after the recombinant adenovirus transfected HUMSCs. Results The recombinant adenovirus Ad-hIFN-γ were constructed successfully, and amplified with titer of 1.6×1010 IU/mL. The titer of Ad-mIFN-γ was 1.0×1010 IU/mL and the titer of Ad-GFP was 1.0×109IU/mL. The green fluorescence proteins could be observed under fluorescent microscope in HUMSCs 24 h after transfection and with a stronger degree after 72 h, and IFN-γ expression in HUMSCs were confirmed by Western blot and ELISA. Conclusion Construction and identification of recombinant adenovirus vector of human/mouse IFN-γ and effectively transfection of HUMSCs were successful.

 

Keywords: IFN-γ HUMSCs, Adenovirus vector, Transfection 

 

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Canninga-van Dijk MR, van der Straaten HM. Fijnheer R, et al. Anti-CD20 monoclonal antibody treatment in 6 patients with therapy-refractory chronic graft-versus-host disease. Blood.2004;104(8):2603-2606.

Darling AJ, Boose JA, Spaltro J. Virus assay methods; accuracy and validation. Biologicals. 1998;26(2): 105-109.

Le Blanc K, Rasmusson I, Sundberg B. et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet,2004;363 (9419):1439-1441.

Ringden O. Uzunel M. Rasmusson I. et al. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplation.2006;81 (10) : 1390-1397.

Ianus A, Holz GG, Theise ND. et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest, 2003; 111 ( 6); 843-850.

Lu LL. Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem Cells with hematopoiesis-supportive function and other potentials. Haematologica, 2006 ; 91 (8) s 1017-1026.

Kita K, Gauqlitz GG, Phan TT, et al. Isolation and characterization of mesenchymal stem cells from the sub- amniotic human umbilical cord lining membrane. Stem cells Dev.2009;19(4);491-506.

Frucht DM, Fukao T, Bogdan C, etal. IFN-gamma production by antigen-presenting cells; mechanisms emerge. Trends Immunol,2001 ;22( 10):556-560.

Asavaroengchai W. Wang H. Wang S, etal. An essential role for IFN-gamma in regulation of alloreactive CDS T cells following allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant ,2007 ; 13(1); 46-55.

King NJ, Thomas SR. Molecules in focus; indolemine 2,3- dioxygenase. Int J Biochem Cell Biol,2007;39( 12) ;2167-2172. Puccetti P, Grohmann U. IIX) and regulatory T cells; a role for reverse signaling and non-canonical NF-kappa В activation. Nat Rev Immunol,2007;7( 10);817-823.

Yoo KH, Janq IK, Lee MW, et al. Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol,2009 ;259(2): 150-156.

Alden TD, Pittman DD, Beres EJ, et al. Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy. J Neurosurq, 1999 ;90( 1 Suppl) : 109-114.


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