Skip to main content
Log in

The last intron of the human thrombopoietin gene enhances expression in milk of transgenic mice

  • Original Paper
  • Published:
Functional & Integrative Genomics Aims and scope Submit manuscript

Abstract

Introns can enhance gene expression levels. This effect is known as intron-mediated enhancement, which is different from that of enhancers or promoters. In our previous study, under the control of the cytomegalovirus or goat β-casein promoter, the vector containing intron V-TPOcDNA expressed the highest thrombopoietin (TPO) level, whereas the vector containing TPOgDNA expressed the lowest level. In order to verify whether intron V also improves TPO expression in the milk of transgenic mice, rat whey acidic protein promoter was used as regulatory element to construct mammary gland expression vectors including pTPOWA (containing TPOcDNA), pTPOWB (containing intron V-TPOcDNA), and pTPOWC (containing TPOgDNA). These vectors were transfected into HC-11 cells and the supernatants were analyzed at 48 h. The highest TPO level was found in pTPOWB (795 pg/mL) and the lowest level in pTPOWC (193 pg/mL). Then, corresponding vectors were microinjected into fertilized mice zygotes. Transgenic mice were identified by polymerase chain reaction and Southern blot. Enzyme-linked immunosorbent assay was performed to measure TPO levels in the milk of lactating transgenic mice. The highest and lowest TPO levels were found in transgenic mice carrying intron V-TPOcDNA (2,307 pg/mL) and in transgenic mice carrying TPOgDNA (242 pg/mL), respectively. Thus, intron V remarkably improved TPO expression in transgenic mice.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Archibald AL, McClenaghan M, Hornsey V, Simons JP, Clark AJ (1990) High-level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice. Proc Natl Acad Sci U S A 87:5178–5182

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Buchman AR, Berg P (1988) Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol 8:4395–4405

    PubMed Central  PubMed  CAS  Google Scholar 

  • Buhler TA, Bruyere T, Went DF, Stranzinger G, Burki K (1990) Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits. Biotechnology (N Y) 8:140–143

    Article  CAS  Google Scholar 

  • Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1:1183–1200

    Article  PubMed  CAS  Google Scholar 

  • Cazzola M, Skoda RC (2000) Translational pathophysiology: a novel molecular mechanism of human disease. Blood 95:3280–3288

    PubMed  CAS  Google Scholar 

  • Chang MS, McNinch J, Basu R, Shutter J, Hsu RY, Perkins C, Mar V, Suggs S, Welcher A, Li L et al (1995) Cloning and characterization of the human megakaryocyte growth and development factor (MGDF) gene. J Biol Chem 270:511–514

    Article  PubMed  CAS  Google Scholar 

  • Dame C, Wolber EM, Freitag P, Hofmann D, Bartmann P, Fandrey J (2003) Thrombopoietin gene expression in the developing human central nervous system. Brain Res Dev Brain Res 143:217–223

    Article  PubMed  CAS  Google Scholar 

  • de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ et al (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369:533–538

    Article  PubMed  Google Scholar 

  • Duncker BP, Davies PL, Walker VK (1997) Introns boost transgene expression in Drosophila melanogaster. Mol Gen Genet 254:291–296

    Article  PubMed  CAS  Google Scholar 

  • Farese AM, Hunt P, Boone T, MacVittie TJ (1995) Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates. Blood 86:54–59

    PubMed  CAS  Google Scholar 

  • Ghilardi N, Skoda RC (1999) A single-base deletion in the thrombopoietin (TPO) gene causes familial essential thrombocythemia through a mechanism of more efficient translation of TPO mRNA. Blood 94:1480–1482

    PubMed  CAS  Google Scholar 

  • Ghilardi N, Wiestner A, Skoda RC (1998) Thrombopoietin production is inhibited by a translational mechanism. Blood 92:4023–4030

    PubMed  CAS  Google Scholar 

  • Ghilardi N, Wiestner A, Kikuchi M, Ohsaka A, Skoda RC (1999) Hereditary thrombocythaemia in a Japanese family is caused by a novel point mutation in the thrombopoietin gene. Br J Haematol 107:310–316

    Article  PubMed  CAS  Google Scholar 

  • Gordon K, Lee E, Vitale JA, Smith AE, Westphal H, Hennighausen L (1987) Production of human tissue plasminogen activator in transgenic mouse milk. Biotechnology 24:425–428

    Google Scholar 

  • Gurney AL, Kuang WJ, Xie MH, Malloy BE, Eaton DL, de Sauvage FJ (1995) Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin. Blood 85:981–988

    PubMed  CAS  Google Scholar 

  • Hokom MM, Lacey D, Kinstler OB, Choi E, Kaufman S, Faust J, Rowan C, Dwyer E, Nichol JL, Grasel T et al (1995) Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice. Blood 86:4486–4492

    PubMed  CAS  Google Scholar 

  • Hoshi S, Yoshitomi H, Komatsu N, Yoshitake S, Okada M (1997) Megakaryocytopoietic activity of a truncated variant of mouse thrombopoietin. Biochem Biophys Res Commun 231:823–826

    Article  PubMed  CAS  Google Scholar 

  • Kamura T, Handa H, Hamasaki N, Kitajima S (1997) Characterization of the human thrombopoietin gene promoter. A possible role of an Ets transcription factor, E4TF1/GABP. J Biol Chem 272:11361–11368

    Article  PubMed  CAS  Google Scholar 

  • Kaushansky K, Broudy VC, Lin N, Jorgensen MJ, McCarty J, Fox N, Zucker-Franklin D, Lofton-Day C (1995) Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development. Proc Natl Acad Sci U S A 92:3234–3238

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kuter DJ (1996) Thrombopoietin: biology and clinical applications. Oncologist 1:98–106

    PubMed  CAS  Google Scholar 

  • Kuter DJ (2010) Biology and chemistry of thrombopoietic agents. Semin Hematol 47:243–248

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW (1990) Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol 15:913–920

    Article  PubMed  CAS  Google Scholar 

  • McCarty JM, Sprugel KH, Fox NE, Sabath DE, Kaushansky K (1995) Murine thrombopoietin mRNA levels are modulated by platelet count. Blood 86:3668–3675

    PubMed  CAS  Google Scholar 

  • Min W, Woo HJ, Lee CS, Lee KK, Yoon WK, Park HW, Kim MH (1998) 307-bp fragment in HOXA7 upstream sequence is sufficient for anterior boundary formation. DNA Cell Biol 17:293–299

    Article  PubMed  CAS  Google Scholar 

  • Murray LJ, Luens KM, Estrada MF, Bruno E, Hoffman R, Cohen RL, Ashby MA, Vadhan-Raj S (1998) Thrombopoietin mobilizes CD34+ cell subsets into peripheral blood and expands multilineage progenitors in bone marrow of cancer patients with normal hematopoiesis. Exp Hematol 26:207–216

    PubMed  CAS  Google Scholar 

  • Ning YS, Zhou HW, Zhou MQ, Chen ZH, Fang XD, Fu N, Wang XN (2001) Cloning and sequence analysis of cDNA and genomic DNA of human thrombopoietin gene. Di Yi Jun Yi Da Xue Xue Bao 21:881–884

    PubMed  CAS  Google Scholar 

  • Ning YS, Li Y, Zhou MQ, Li M, Zeng YT, Wang XN (2004) Role of intron and 5′ untranslated region in human thrombopoietin gene expression. Di Yi Jun Yi Da Xue Xue Bao 24:991–994

    PubMed  CAS  Google Scholar 

  • Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster RL (1991) Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci U S A 88:478–482

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Parra G, Bradnam K, Rose AB, Korf I (2011) Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res 39:5328–5337

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Sungaran R, Markovic B, Chong BH (1997) Localization and regulation of thrombopoietin mRNa expression in human kidney, liver, bone marrow, and spleen using in situ hybridization. Blood 89:101–107

    PubMed  CAS  Google Scholar 

  • Tahara T, Usuki K, Sato H, Ohashi H, Morita H, Tsumura H, Matsumoto A, Miyazaki H, Urabe A, Kato T (1996) A sensitive sandwich ELISA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders. Br J Haematol 93:783–788

    Article  PubMed  CAS  Google Scholar 

  • Velander WH, Page RL, Morcol T, Russell CG, Canseco R, Young JM, Drohan WN, Gwazdauskas FC, Wilkins TD, Johnson JL (1992) Production of biologically active human protein C in the milk of transgenic mice. Ann N Y Acad Sci 665:391–403

    Article  PubMed  CAS  Google Scholar 

  • Wolber EM, Dame C, Fahnenstich H, Hofmann D, Bartmann P, Jelkmann W, Fandrey J (1999) Expression of the thrombopoietin gene in human fetal and neonatal tissues. Blood 94:97–105

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The research was supported by “1035” key research project of Chinese National Science and Technology Committee (no. 96-901-009).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yunshan Ning.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Y., Zhou, M., Zhou, H. et al. The last intron of the human thrombopoietin gene enhances expression in milk of transgenic mice. Funct Integr Genomics 14, 229–236 (2014). https://doi.org/10.1007/s10142-013-0348-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10142-013-0348-x

Keywords

Navigation