Biopolym. Cell. 2007; 23(2):100-107.
Molecular and Cell Biotechnologies
Plasmid vector for the human preproinsuiin gene delivery into mammalian cells
1Toporova O. K., 1Kyrylenko S. D., 1Irodov D. M., 1Kordium V. A.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


Eukaryotic expression vector for human preproinsuiin gene has been constructed for further delivery into non-endocrine mammalian cells in vitro and in vivo for the development of experimental gene therapy for type I diabetes. Vector construction consists of two independent modules, namely, bacterial plasmid sequence that allows its replication in Escherichia coli cells, and expression cassette for the target and marker genes which is flanked by inverted terminal repeats of human adeno-associated virus. The human cytomegalovirus immediate-early promoter has been used to ensure the constitutive expression of human preproinsuiin gene. The hepatitis B viral enhancer 1 was subcloned into cassette to increase the target transgene expression. After vector construction in vitro transfer into mammalian cells of different origin, the stable transformants were obtained and the availability of target human preproinsuiin transgene in the transformed cells has been confirmed by PCR analysis.
Keywords: human preproinsuiin gene, expression vector, stable transformants


[1] Strauss M, Barranger JA. Concepts in gene therapy. Berlin: DeGruyter, 1997. 553 p.
[2] Hodgson CP. The vector void in gene therapy. Biotechnology (N Y). 1995;13(3):222-5.
[3] Relph K, Harrington K, Pandha H. Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ. 2004;329(7470):839-42.
[4] Sandmair AM, Loimas S, Puranen P, Immonen A, Kossila M, Puranen M, Hurskainen H, Tyynel? K, Turunen M, Vanninen R, Lehtolainen P, Palj?rvi L, Johansson R, Vapalahti M, Yl?-Herttuala S. Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses. Hum Gene Ther. 2000;11(16):2197-205.
[5] St George JA. Gene therapy progress and prospects: adenoviral vectors. Gene Ther. 2003;10(14):1135-41. PubMed PMID: 11050953.
[6] Hitt MM, Graham FL. Adenovirus vectors for human gene therapy. Adv Virus Res. 2000;55:479-505.
[7] Schr?der AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002;110(4):521-9.
[8] Schagen FH, Ossevoort M, Toes RE, Hoeben RC. Immune responses against adenoviral vectors and their transgene products: a review of strategies for evasion. Crit Rev Oncol Hematol. 2004;50(1):51-70.
[9] Muruve DA. The innate immune response to adenovirus vectors. Hum Gene Ther. 2004;15(12):1157-66.
[10] Smith TA, Idamakanti N, Rollence ML, Marshall-Neff J, Kim J, Mulgrew K, Nemerow GR, Kaleko M, Stevenson SC. Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum Gene Ther. 2003;14(8):777-87.
[11] Smith JS, Tian J, Lozier JN, Byrnes AP. Severe pulmonary pathology after intravenous administration of vectors in cirrhotic rats. Mol Ther. 2004;9(6):932-41.
[12] 12. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, de Saint Basile G, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, Cavazzana-Calvo M. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003;302(5644):415-9.
[13] Kordium VA, Toporova OK, Novikova SM, Lihacheva LI, Suhorada OM, Ruban TA, Kozel JO, Potapenko RI, Irodov DM. Development approaches to gene therapy of atherosclerosis. Zh Akad Med Nayk Ukrainy. 2004; 10(2): 288-300.
[14] Toporova OK, Novikova SN, Lihacheva LI, Suhorada OM, Ruban TA, Kozel JA, Irodov DM, Kordium VA. Non-viral gene delivery of human apoA1 into mammalian cells in vitro and in vivo. Biopolym Cell. 2004; 20(1-2):25-32.
[15] Sambrook J, Fritsch E, Maniatis T. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Lab. press, 1989.
[16] Stoll SM, Calos MP. Extrachromosomal plasmid vectors for gene therapy. Curr Opin Mol Ther. 2002;4(4):299-305.
[17] Gromnatskiy NI. Diabetology. M.: GOU VUNMC MZ RF, 2002. 254 p.
[18] Yang CC, Xiao X, Zhu X, Ansardi DC, Epstein ND, Frey MR, Matera AG, Samulski RJ. Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro. J Virol. 1997;71(12):9231-47.
[19] H?ser D, Heilbronn R. Adeno-associated virus integrates site-specifically into human chromosome 19 in either orientation and with equal kinetics and frequency. J Gen Virol. 2003;84(Pt 1):133-7.
[20] Neznanov NS, Troianovski? BM, Gartel' AL, Makarova IV, Bendukidze KA. [Molecular cloning of the human insulin gene]. Mol Gen Mikrobiol Virusol. 1987;(7):14-5.
[21] Lukash LL, Podolskaya SV, Neborachko LN, Sukhorada HM, Varzanova IS, Kordyum VA. Expression of insulin gene introduced into embryonic human cells. Advances in gene technology: Molecular biology of human genetic disease: Proc. Miami Bio. Technol Eur Symp. (Short Reports.) Monaco, 1994. Vol. 5: 62.