ABSTRACT
Background: Cancer/Testis Antigens [CTAs] are a subgroup 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
ABSTRACT
Autosomal recessive spinal muscular atrophy is a disease resulting from homozygous absence of SMN1 gene in approximately 94% of SMA patients. To identify patients who retained a single SMN1 copy, SMN1 dosage analysis was performed by quantitative Real-time PCR using SYBR green dye. SMN1 dosage analysis results were utilized to identify carriers before offering prenatal diagnosis. Carrier testing was performed for 150 individuals. Copy number of the SMN1 gene was determined by the comparative threshold cycle [Ct] method and human serum albumin gene was used as a reference. Analysis of 150 DNA samples with quantitative PCR determined the number of SMN1 gene copies. Of these, 50 [33.33%] cases had one SMN1 gene copy, 87 [58%] had two copies and 13 [8.66%] did not have any copies of SMN1. The homozygous SMN1 deletion ratio was 0.00 and deletion of one copy of SMN1 gene ratio ranged from 0.3 to 0.58. This report demonstrates modification of risk estimation for the diagnosis and detection of SMA carriers by accurate determination of SMN1 copy number. SMN1 copy number analysis is an important parameter for identification of couples at risk of having children affected with SMA. It also reduces unwarranted prenatal diagnosis for SMA. Furthermore, the dosage analysis might be useful for the counseling of clinically suspected SMA patients with negative diagnostic SMA tests