Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jul;9(7):255.
doi: 10.1007/s13205-019-1786-5. Epub 2019 Jun 7.

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

Fatemeh Farzaneh  1 , Zohreh Mirzapoor  1 , Ehsan Jahangirian  1 , Farid Heidari  1 , Ehsan Hashemi  1 , Aidin Rahim-Tayefeh  1 , Nayeralsadat Fatemi  2 , Abbas Jamshidizad  1 , Mojtaba Dashtizad  1 , Mehdi Shamsara  1
Affiliations

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

Fatemeh Farzaneh et al. 3 Biotech. 2019 Jul.

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.

Keywords: Chromosomal position effect; Pgk-1 promoter; Transgenic mouse; cHS4 insulator.

PubMed Disclaimer

Conflict of interest statement

Conflict of interestThe authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic representation of the cassettes and its in vitro assessment. a INPGK-EGFP cassette was designed by replacing the CMV promoter in the CMV-EGFP cassette with the Pgk-1 promoter. After that, three copies of cHS4 insulators amplified from hen genome were inserted at the sides of the cassette. b Transient transfection of the two cassettes, including CMV-EGFP as a control and INPGK-EGFP, separately into HEK293T cells confirmed the capability of the engineered cassette in expression of the EGFP protein by a fluorescent microscope
Fig. 2
Fig. 2
Transgenic mice generated by injection of the designed cassette. a Transgenic founders were identified by PCR on tail-biopsies extracted DNA. The Taf3 endogenous sequence was amplified to examine the PCR compatibility of the extracted DNA. b Transgenic mice produced by the pronuclear injection showed the different intensities of fluorescent signal
Fig. 3
Fig. 3
Investigation of the transgene copy-number-dependent intensity of the fluorescent signal. a Measurement of the EGFP fluorescence in the culture of fibroblast cells derived from the transgenic F1 offspring (TG) and wild-type (WT) mice. b Determination of relative gene copy number in the fibroblasts by real-time PCR. The data showed no correlation between the EGFP fluorescent signal and gene copy number. All experiments were performed in three independent experiments and the results were presented as Mean ± SD
Fig. 4
Fig. 4
Visual observation of the fluorescent signal heterogeneity in the F1 transgenic mouse organs. a Heart, b liver, c kidney, and d spleen
See this image and copyright information in PMC

References

    1. Aker M, Tubb J, Groth AC, Bukovsky AA, Bell AC, Felsenfeld G, Kiem HP, Stamatoyannopoulos G, Emery DW. Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects. Hum Gene Ther. 2007;18:333–343. doi: 10.1089/hum.2007.021. - DOI - PubMed
    1. Chung JH, Whiteley M, Felsenfeld G. 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. 1993;74:505–514. doi: 10.1016/0092-8674(93)80052-G. - DOI - PubMed
    1. Emery DW, Yannaki E, Tubb J, Stamatoyannopoulos G. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc Natl Acad Sci USA. 2000;97:9150–9155. doi: 10.1073/pnas.160159597. - DOI - PMC - PubMed
    1. Emery DW, Aker M, Stamatoyannopoulos G. Chromatin insulators and position effects. New Compr Biochem. 2003;38:381–395. doi: 10.1016/S0167-7306(03)38023-8. - DOI
    1. Frazar TF, Weisbein JL, Anderson SM, Cline AP, Garrett LJ, Felsenfeld G, Gallagher PG, Bodine DM. 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. 2003;23:4753–4763. doi: 10.1128/MCB.23.14.4753-4763.2003. - DOI - PMC - PubMed

LinkOut - more resources