Skip to main content

Advertisement

SpringerLink
Log in

The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

Abstract

Differential expression of transgenes in transgenic animals is one of the main drawbacks of pronuclear injection. To overwhelm this issue, the genetic constructs are equipped with insulators. In this study, the consensus of exerting chicken hypersensitive site-4 (cHS4) insulator was examined on the shield of phosphoglycerate kinase-1 (Pgk-1) promoter from the surrounding chromatin in transgenic mice. The PGK-EGFP cassette was flanked by insertion of three copies of the cHS4 insulators. Mouse zygotes’ microinjection by the constructed cassette was resulted in the birth of nine transgenic founders (F0). Copy-number-dependent expression of the EGFP was investigated in the transgenic F1 offspring by fluorometry and real-time PCR. They showed no correlation between the expression level of transgene and gene copy number among the transgenic lines. Moreover, dissection of the EGFP-expressing mice revealed heterogeneous expression of the EGFP in the different organs. In conclusion, for the first time, these findings indicated that the cHS4 sequence is not a perfect insulator to fully protect the Pgk-1 promoter from the side effects of integration site in transgenic mice and it needs probably to some additional elements of the cHS4 locus to reach this goal.

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

Access this article

Log in via an institution

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

  • Aker M, Tubb J, Groth AC, Bukovsky AA, Bell AC, Felsenfeld G, Kiem HP, Stamatoyannopoulos G, Emery DW (2007) Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther 18:333–343

    Article  CAS  Google Scholar 

  • Chung JH, Whiteley M, Felsenfeld G (1993) A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74:505–514

    Article  CAS  Google Scholar 

  • Emery DW, Yannaki E, Tubb J, Stamatoyannopoulos G (2000) A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA 97:9150–9155

    Article  CAS  Google Scholar 

  • Emery DW, Aker M, Stamatoyannopoulos G (2003) Chromatin insulators and position effects. New Compr Biochem 38:381–395

    Article  CAS  Google Scholar 

  • Frazar TF, Weisbein JL, Anderson SM, Cline AP, Garrett LJ, Felsenfeld G, Gallagher PG, Bodine DM (2003) Variegated expression from the murine band 3 (AE1) promoter in transgenic mice is associated with mRNA transcript initiation at upstream start sites and can be suppressed by the addition of the chicken beta-globin 5′ HS4 insulator element. Mol Cell Biol 23:4753–4763

    Article  CAS  Google Scholar 

  • Giraldo P, Rival-Gervier S, Houdebine LM, Montoliu L (2003) The potential benefits of insulators on heterologous constructs in transgenic animals. Transgenic Res 12:751–755

    Article  CAS  Google Scholar 

  • Grajevskaja V, Balciuniene J, Balciunas D (2013) Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish. Mol Genet Genom 288:717–725

    Article  CAS  Google Scholar 

  • Hamaguchi I, Woods NB, Panagopoulos I, Andersson E, Mikkola H, Fahlman C, Zufferey R, Carlsson L, Trono D, Karlsson S (2000) Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro. J Virol 74:10778–10784

    Article  CAS  Google Scholar 

  • Haruyama N, Cho A, Kulkarni AB (2009) Overview: engineering transgenic constructs and mice. Curr Protoc Cell Biol. https://doi.org/10.1002/0471143030.cb1910s42

    Article  PubMed  PubMed Central  Google Scholar 

  • Hickman-Davis JM, Davis IC (2006) Transgenic mice. Paediatr Respir Rev 7:49–53

    Article  Google Scholar 

  • Kingston RE, Chen CA, Rose JK (2003) Calcium phosphate transfection. Curr Protoc Mol Biol. https://doi.org/10.1002/0471142727.mb0901s63

    Article  PubMed  Google Scholar 

  • Martin DI, Whitelaw E (1996) The vagaries of variegating transgenes. BioEssays 18:919–923

    Article  CAS  Google Scholar 

  • McBurney MW, Staines WA, Boekelheide K, Parry D, Jardine K, Pickavance L (1994) Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice. Dev Dyn 200:278–293

    Article  CAS  Google Scholar 

  • Moltó E, Vicente-García C, Fernández A, Montoliu L (2011) Genomic insulators in transgenic animals. In: Brakebusch C, Pihlajaniemi T (eds) Mouse as a model organism. Springer, Dordrecht, pp 1–10

    Google Scholar 

  • Pikaart MJ, Recillas-Targa F, Felsenfeld G (1998) Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 12:2852–2862

    Article  CAS  Google Scholar 

  • Potts W, Tucker D, Wood H, Martin C (2000) Chicken beta-globin 5′ HS4 insulators function to reduce variability in transgenic founder mice. Biochem Biophys Res Commun 273:1015–1018

    Article  CAS  Google Scholar 

  • Recillas-Targa F, Valadez-Graham V, Farrell CM (2004) Prospects and implications of using chromatin insulators in gene therapy and transgenesis. BioEssays 26:796–807

    Article  CAS  Google Scholar 

  • Rincón-Arano H, Furlan-Magaril M, Recillas-Targa F (2007) Protection against telomeric position effects by the chicken cHS4 beta-globin insulator. Proc Natl Acad Sci USA 104:14044–14049

    Article  Google Scholar 

  • Rivella S, Callegari JA, May C, Tan CW, Sadelain M (2000) The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J Virol 74:4679–4687

    Article  CAS  Google Scholar 

  • Taboit-Dameron F, Malassagne B, Viglietta C, Puissant C, Leroux-Coyau M, Chéreau C, Attal J, Weill B, Houdebine LM (1999) Association of the 5′ HS4 sequence of the chicken beta-globin locus control region with human EF1 alpha gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits. Transgenic Res 8:223–235

    Article  CAS  Google Scholar 

  • Wang Y, DeMayo FJ, Tsai SY, O’Malley BW (1997) Ligand-inducible and liver-specific target gene expression in transgenic mice. Nat Biotechnol 15:239–243

    Article  CAS  Google Scholar 

  • Whitelaw CBA, Springbett AJ, Webster J, Clark J (1993) The majority of G0 transgenic mice are derived from mosaic embryos. Transgenic Res 2:29–32

    Article  CAS  Google Scholar 

  • Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6:679–714

    Article  CAS  Google Scholar 

  • Yannaki E, Tubb J, Aker M, Stamatoyannopoulos G, Emery DW (2002) Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors. Mol Ther 5:589–598

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The research was funded by the Iran National Science Foundation (INSF) (Grant Number 91001148). The authors thank the National Institute of Genetic Engineering and Biotechnology for instrumental supports.

Author information

Authors and Affiliations

  1. National Research Center for Transgenic Mouse, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, Iran

    Fatemeh Farzaneh, Zohreh Mirzapoor, Ehsan Jahangirian, Farid Heidari, Ehsan Hashemi, Aidin Rahim-Tayefeh, Abbas Jamshidizad, Mojtaba Dashtizad & Mehdi Shamsara

  2. Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, P.O. Box 19395-4644, Tehran, Iran

    Nayeralsadat Fatemi

Authors
  1. Fatemeh Farzaneh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zohreh Mirzapoor
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ehsan Jahangirian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Farid Heidari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ehsan Hashemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aidin Rahim-Tayefeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nayeralsadat Fatemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Abbas Jamshidizad
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mojtaba Dashtizad
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mehdi Shamsara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mehdi Shamsara.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Farzaneh, F., Mirzapoor, Z., Jahangirian, E. et al. The chicken hypersensitive site-4 insulator cannot fully shield the murine phosphoglycerate kinase-1 promoter from integration site effects in transgenic mice. 3 Biotech 9, 255 (2019). https://doi.org/10.1007/s13205-019-1786-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-019-1786-5

Keywords

Search

Navigation