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Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression in Esophageal Squamous Carcinoma Cell Line KYSE-30

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Abstract

Gene delivery to esophageal tissue could provide novel treatments for diseases, such as cancer. The Sleeping Beauty (SB) transposon system, as a natural and non-viral tool, is efficient at transferring transgene into the human genome for human cell genetic engineering. The plasmid-based SB transposon can insert into chromosomes through an accurate recombinase-mediated mechanism, providing long-term expression of transgene integrated into the target cells. In this study, we aimed to investigate the activity of ED-L2 tissue-specific promoter that was engineered from the Epstein-Barr Virus (EBV) and combined with the hyperactive SB100X transposase to achieve the stable expression of T2-Onc3 transposon in esophageal squamous epithelial cells. Here we constructed an SB transposon-based plasmid system to obtain the stable expression of transposon upon introduction of a hyperactive SB transposase under the control of tissue-specific ED-L2 promoter via the lipid-based delivery method in the cultured esophageal squamous cell carcinoma cells. Among established human and mouse cell lines, the (ED-L2)-SB100X transposase was active only in human esophageal stratified squamous epithelial and differentiated keratinocytes derived from skin (KYSE-30 and HaCaT cell lines), where it revealed high promoter activity. Data offered that the 782 bp sequence of ED-L2 promoter has a key role in its activity in vitro. The (ED-L2)-SB100X transposase mediated stable integration of T2-Onc3 in KYSE-30 cells, thereby providing further evidence of the tissue specificity of ED-L2 promoter. The KYSE-30 cells modified with the SB system integrate on average 187 copies of the T2-Onc3 transposon in its genome. In aggregate, the (ED-L2)-SB100X transposase can be efficiently applied for the tissue-specific stable expression of a transgene in human KYSE-30 cells using SB transposon.

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Data Availability

Data will be available on request.

Abbreviations

SB:

Sleeping Beauty

iPSC:

Induced pluripotent stem cell

GEM:

Genetically engineered model

PB:

PiggyBac

IRs:

Inverted repeats

TSP:

Tissue-specific promoter

EBV:

Epstein-Barr virus

PMA:

Phorbol 12-myristate 13-acetate

gDNA:

Genomic DNA

Ct:

Cycle threshold

ALB:

Albumin

MFI:

Mean florescence intensity

CAG:

CMV enhancer/chicken beta-actin promoter

TF:

Transcription factors

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Acknowledgements

Authors wish to thank the personnel of Division of Human Genetics of Avicenna Research Institute and ACECR-Khorasan Razavi Branch for kindest collaboration.

Funding

The results presented in this paper were part of a PhD student thesis that was supported by Mashhad University of Medical Sciences (# 921706).

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Author notes

  1. Mohammad Reza Abbaszadegan and Moein Farshchian have contributed equally to this work.

Authors and Affiliations

  1. Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Reihaneh Alsadat Mahmoudian

  2. Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran

    Fardin Fathi

  3. Stem Cell and Regenerative Medicine Research Group, Iranian Academic Center for Education, Culture and Research (ACECR) Razavi Khorasan, ACECR Central Building, Ferdowsi University Campus, Mashhad- Azadi Square, Mashhad Branch, Mashhad, Iran

    Moein Farshchian

  4. Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

    Mohammad Reza Abbaszadegan

  5. Medical Genetics Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran

    Mohammad Reza Abbaszadegan

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  1. Reihaneh Alsadat Mahmoudian
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  2. Fardin Fathi
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  3. Moein Farshchian
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  4. Mohammad Reza Abbaszadegan
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Correspondence to Moein Farshchian or Mohammad Reza Abbaszadegan.

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Mahmoudian, R.A., Fathi, F., Farshchian, M. et al. Construction and Quantitative Evaluation of a Tissue-Specific Sleeping Beauty by EDL2-Specific Transposase Expression in Esophageal Squamous Carcinoma Cell Line KYSE-30. Mol Biotechnol 65, 350–360 (2023). https://doi.org/10.1007/s12033-022-00490-4

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