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Molecular Biology

, Volume 52, Issue 3, pp 430–435 | Cite as

Insertion of Multiple Artificial Introns of Universal Design into CDNA during Minigene Construction Assures Correct Transgene Splicing

  • M. V. Shepelev
  • M. V. Tikhonov
  • S. V. Kalinichenko
  • I. V. Korobko
Molecular Cell Biology

Abstract

The presence of introns is often required for efficient transgene expression. The use of full-length genes for transgenesis is associated with technical difficulties due to the large size of the genetic construct. To solve this problem, we recently suggested a universal design of small artificial introns that ensures efficient splicing. However, the insertion of more than one intron into cDNA might result in the aberrant splicing of the minigene with exon skipping. Here, we showed that the insertion of two artificial introns of universal design into cDNA resulted in a splicing pattern that corresponds to the excision of each intron with an exon between them remaining in the transcript. No transcript formation with exon skipping was detected. Therefore, the developed design of small artificial introns assures splicing solely between the donor and the acceptor splice sites of each single intron and results in the generation of a correct transcript from minigene premRNA. These findings enable the construction of minigenes for transgenesis with more than one artificial intron, with no additional cis-elements required to prevent aberrant splicing.

Keywords

artificial intron aberrant splicing exon skipping cDNA transgene expression EGFP 

Abbreviation

EGFP

enhanced green fluorescent protein

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References

  1. 1.
    Lugones L.G., Scholtmeijer K., Klootwijk R., Wessels J.G. 1999. Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol. Microbiol. 32, 681–689.CrossRefPubMedGoogle Scholar
  2. 2.
    Gallegos J.E., Rose A.B. 2015. The enduring mystery of intron-mediated enhancement. Plant Sci. 237, 8–15.CrossRefPubMedGoogle Scholar
  3. 3.
    Haddad-Mashadrizeh A., Zomorodipour A., Izadpanah M., Sam M.R., Ataei F., Sabouni F., Hosseini S.J. 2009. A systematic study of the function of the human beta-globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells. J. Gene Med. 11, 941–950.CrossRefPubMedGoogle Scholar
  4. 4.
    Zieler H., Huynh C.Q. 2002. Intron-dependent stimulation of marker gene expression in cultured insect cells. Insect Mol. Biol. 11, 87–95.CrossRefPubMedGoogle Scholar
  5. 5.
    Malik A.K., Wang J.M., Kurachi K. 2001. Effects of a second intron on recombinant MFG retroviral vector. Arch. Virol. 146, 601–609.CrossRefPubMedGoogle Scholar
  6. 6.
    Hural J.A., Kwan M., Henkel G., Hock M.B., Brown M.A. 2000. An intron transcriptional enhancer element regulates IL-4 gene locus accessibility in mast cells. J. Immunol. 165, 3239–3249.CrossRefPubMedGoogle Scholar
  7. 7.
    Le Hir H., Saulière J., Wang Z. 2016. The exon junction complex as a node of post-transcriptional networks. Nat. Rev. Mol. Cell. Biol. 17, 41–54.CrossRefPubMedGoogle Scholar
  8. 8.
    Whitelaw C.B., Archibald A.L., Harris S., McClenaghan M., Simons J.P., Clark A.J. 1991. Targeting expression to the mammary gland: Intronic sequences can enhance the efficiency of gene expression in transgenic mice. Transgenic Res. 1, 3–13.CrossRefPubMedGoogle Scholar
  9. 9.
    Technical Bulletin. 2009. pCI and pSI Mammalian Expression Vectors. Madison, WI: Promega Corp.Google Scholar
  10. 10.
    Tikhonov M.V., Maksimenko O.G., Georgiev P.G., Korobko I.V. 2017. Optimal artificial mini-introns for transgenic expression in the cells of mice and hamsters. Mol. Biol. (Moscow). 51, 592–595.CrossRefGoogle Scholar
  11. 11.
    Gao K., Masuda A., Matsuura T., Ohno K. 2008. Human branch point consensus sequence is yUnAy. Nucleic Acids Res. 36, 2257–2267.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Shepelev M.V., Chernoff J., Korobko I.V. 2011. Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation. Small GTPases. 2, 17–26.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Brinster R.L., Allen J.M., Behringer R.R., Gelinas R.E., Palmiter R.D. 1988. Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 85, 836–840.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • M. V. Shepelev
    • 1
  • M. V. Tikhonov
    • 1
  • S. V. Kalinichenko
    • 1
  • I. V. Korobko
    • 1
  1. 1.Institute of Gene BiologyRussian Academy of SciencesMoscowRussia

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