Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System

Maryam Khaleghizadeh; Mohammad Mahdi Forghanifard; Abolfazl Rad; Moein Farshchian; Zahra Hejazi; Mehran Gholamin; Bahram Memar; Mohammad Reza Abbaszadegan

Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System

Avicenna J Med Biotechnol. 2018 Apr-Jun;10(2):75-82.

Authors

Maryam Khaleghizadeh^{1

2}, Mohammad Mahdi Forghanifard³, Abolfazl Rad⁴, Moein Farshchian², Zahra Hejazi¹, Mehran Gholamin², Bahram Memar⁵, Mohammad Reza Abbaszadegan^{2

6}

Affiliations

¹ Department of Microbiology, Damghan Branch, Islamic Azad University, Damghan, Iran.
² Human Genetic Division, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
³ Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.
⁴ Cellular and Molecular Research Center, Sabzevar University of Medical Sciences, Sabzevar, Iran.
⁵ Department of Pathology, Imam Reza Hospital, Mashhad University of Medical Sciences, Mashhad, Iran.
⁶ Medical Genetics Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

PMID: 29849983
PMCID: PMC5960063

Abstract

Background: Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary.

Methods: In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2 gene by real-time PCR.

Results: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of DPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression.

Conclusion: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells in vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

Keywords: Carcinogenesis; Esophageal squamous cell carcinoma; Germ cells; Testis.