Investigation in vitro expression of catSper sub fragment followed by production of polyclonal antibody: potential candidate for the next generation of non hormonal contraceptive

CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper [1-4] ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive. In this experimental study, a small extracellular fragment of Cat-Sper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1] pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2] The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay [ELISA] and western-blot, and 3] The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test. The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with [p< 0.05] unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility. CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive

[Inhibition of ackA and pta genes using two specific antisense RNAs reduced acetate accumulation in batch fermentation of E. coli BL21 [DE3]]

Expression of foreign proteins in E. coli is normally inhibited by exogenous production of acetate. To overcome this problem, various strategies have been proposed and tested to reduce the extent of acetate accumulation. Although these strategies can improve the outcome, the implementation of their proposed techniques is not practical. Because to achieve optimal results, it requires extremely tight control conditions and the actual cost is very high. Furthermore, a simple knockout mutation of the target metabolic pathway would not be appropriate because the acetate pathway plays an important physiological role in E. coli. In this study, we employed an antisense RNA strategy as an elaborated metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, acetate kinase [ACK] and phosphotransacetylase [PTA]. The fragments of antisense cassette were cloned sequentially in pBluescriptsk+ and completed cassette subcloned in pLT10T3. The function of this cassette was evaluated with RT-PCR and ACK and PTA assay. The effect of cassette on cell physiology was monitored by determination of optical density, glucose consumption and acetate production. We found that the antisense method partially reduced mRNA levels of the target genes, lowered the concentration of acetate in culture media and increased growth rate and final cell density in antisense-regulated strain. This strategy could provide us with a useful, inexpensive and practical tool to achieve a large-scale protein production system

effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line

The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment

Wild type p53 gene transfer increases chemosensitivity and apoptotic response of PANC-1 pancreatic tumor cell line

The effect of p53 gene therapy on chemosensitivity and apoptotic response of PANC-1 tumor cells, which express high amount of mutant p53, to cancer chemotherapeutic agents of Etoposide and Doxorubicin was investigated. Comparison of the chemosensitivity of PANC-1 cells to its wild type p53 transfectants showed that wt-p53 expressing transfectants are more sensitive to both Etoposide and Doxorubucin. It further showed that neither the PANC1 cells nor its wild type p53 transfectants arrested at G1 in response toX-irradiation. Furthermore, treatment of both PANC-1 cells as well as its wt-p53 transfectants with etoposide resulted in apoptosis despite the difference in their p53 status, although, the number of apoptotic cells of the wt-p53 transfectants was higher compared to the control cells. This evidence reinforce the view that combining p53 gene therapy with conventional chemotherapeutic agents may yield a more beneficial response than conventional treatments alone in pancreatic tumor cells with high amount of mutant p53