Molecular Biology

, Volume 53, Issue 3, pp 362–370 | Cite as

Human Antithrombin III Minigene with an Optimized Splicing Pattern

  • M. V. ShepelevEmail author
  • E. K. Saakian
  • S. V. Kalinichenko
  • I. V. Korobko


Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.


minigene antithrombin III splicing pattern aberrant exon alternative transcript 



  1. 1.
    Luxembourg B., Delev D., Geisen C., Spannagl M., Krause M., Miesbach W., Heller C., Bergmann F., Schmeink U., Grossmann R., Lindhoff-Last E., Seifried E., Oldenburg J., Pavlova A. 2011. Molecular basis of antithrombin deficiency. Thromb. Haemost. 105, 635–646.CrossRefGoogle Scholar
  2. 2.
    Konkle B.A., Bauer K.A., Weinstein R., Greist A., Holmes H.E., Bonfiglio J. 2003. Use of recombinant human antithrombin in patients with congenital antithrombin deficiency undergoing surgical procedures. Transfusion. 43, 390–394.CrossRefGoogle Scholar
  3. 3.
    Paidas M.J., Triche E.W., James A.H., DeSancho M., Robinson C., Lazarchick J., Ornaghi S., Frieling J. 2016. Recombinant human antithrombin in pregnant patients with hereditary antithrombin deficiency: Integrated analysis of clinical data. Am. J. Perinatol. 33, 343–349.Google Scholar
  4. 4.
    Shepelev M.V., Kalinichenko S.V., Deykin A.V., Korobko I.V. 2018. Production of recombinant proteins in the milk of transgenic animals: Current state and prospects. Acta Naturae. 10, 40–47.CrossRefGoogle Scholar
  5. 5.
    Lavine G. 2009. FDA approves first biological product derived from transgenic animal. Am. J. Health Syst. Pharm. 66 (6), 518. CrossRefGoogle Scholar
  6. 6.
    Edmunds T., Van Patten S.M., Pollock J., Hanson E., Bernasconi R., Higgins E., Manavalan P., Ziomek C., Meade H., McPherson J.M., Cole E.S. 1998. Transgenically produced human antithrombin: Structural and functional comparison to human plasma-derived antithrombin. Blood. 91, 4561–4571.Google Scholar
  7. 7.
    Palmiter R.D., Sandgren E.P., Avarbock M.R., Allen D.D., Brinster R.L. 1991. Heterologous introns can enhance expression of transgenes in mice. Proc. Natl. Acad. Sci. U. S. A. 88, 478–482.CrossRefGoogle Scholar
  8. 8.
    Choi T., Huang M., Gorman C., Jaenisch R. 1991. A generic intron increases gene expression in transgenic mice. Mol. Cell. Biol. 11, 3070–3074.CrossRefGoogle Scholar
  9. 9.
    Raker V.A., Mironov A.A., Gelfand M.S., Pervouchine D.D. 2009. Modulation of alternative splicing by long-range RNA structures in Drosophila. Nucleic Acids Res. 37, 4533–4544.CrossRefGoogle Scholar
  10. 10.
    Gelfand M.S. 1989. Statistical analysis of mammalian pre-mRNA splicing sites. Nucleic Acids Res. 17, 6369–6382.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2019

Authors and Affiliations

  • M. V. Shepelev
    • 1
    Email author
  • E. K. Saakian
    • 1
  • S. V. Kalinichenko
    • 1
  • I. V. Korobko
    • 1
  1. 1.Institute of Gene Biology, Russian Academy of SciencesMoscowRussia

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