Ectopic expression of human DPPA2 gene in ESCC cell line using retroviral system

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

increase in protein and plasmid yields of E. coli with optimized concentration of ampicillin as selection marker

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid

Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized

Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG [0.5 mM] and incubation with 0, 100, 200 and 300 micro g.mL[-1] ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve

Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 micro g.mL[-1] ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07x10[9], 3.21x10[9], 2.32x10[10] , 8.11x10[8], respectively. The plasmid yields were 55 ng.micro L[-1], 69 ng.micro L[-1], 164 ng.micro L[-1] and 41 ng.micro L[-1], respectively

Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration [200 micro g.mL[-1]] was significantly [p < 0.01] higher than other doses

Real-time PCR: an appropriate approach to confirm ssDNA generation from PCR product in SELEX process

Background: Aptamers are single stranded DNA [ssDNA] or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment [SELEX] method is much dependent on the successful conversion of double stranded DNA [dsDNA] to ssDNA

Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the efficiency of another technique for confirming the ssDNA generation in comparison to the polyacrylamide electrophoresis [PAGE] analysis

Materials and Methods: Real-time PCR was employed in the present study for PCR amplification of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis

Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 [degree]C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds

Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation