Construction of an antisense RNA recombinant adenovirus vector of the human PDE5A1 gene promoter / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct an antisense RNA recombinant adenovirus vector of the human PDE5A1 promoter gene.</p><p><b>METHODS</b>A cDNA fragment containing the human PDE5A1 promoter and the human PDE5A1-specific exon was determined according to the gene bank. The antisense RNA fragment was synthesized artificially and subcloned into the pENTR. Then, the sequence of pENTR fragment was detected, and the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 was constructed by LR reaction with the Gateway expression system. The identified recombinant adenovirus plasmid was digested with Pac I and transformed into 293A cells to package recombinant adenovirus particles. The recombinant adenovirus particles were tested with PCR technique and purified after acquisition by CsCl density gradient ultracentrifugation.</p><p><b>RESULTS</b>The sequencing result showed a 145 bp fragment in pENTR, which was proved to be the domain of the antisense RNA fragment. PCR confirmed that the antisense RNA fragment was cloned into the recombinant adenovirus vector pAd/CMV/V5/antisense-PDE5A1 successfully and could infect 293A cells. The titer of virus stocks was up to 10(8) - 10(10)/microl after purification.</p><p><b>CONCLUSION</b>With the Gateway expression system, the culturing and reproduction of 293A cells can reproduce recombinant adenovirus pAd/CMV/V5-DEST successfully, and the recombinant adenovirus vector can meet the need of in vivo gene transfection in laboratory studies.</p>

The effects of antisenes oligodeoxynucleotide on the cyclic nucleotide monophosphates in smooth muscle cells of human corpus cavernosum / 中华男科学杂志

<p><b>OBJECTIVES</b>To investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction.</p><p><b>METHODS</b>PDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection.</p><p><b>RESULTS</b>After transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01).</p><p><b>CONCLUSIONS</b>The PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.</p>