A Rapid Protocol for Screening Genetically Modified Soybeans Based on Positive Plasmid Molecule Containing Multiple Targets / 中国生物化学与分子生物学报

Recently we witness the rising number of genetically modified (GM) soybean (Glycine max) events approved for importing from abroad and developed domestically, so it is urgent to establish a rapid screening protocol that can cover more events with less detection targets and fit the national condition. Additionally, in order to control the detection workload, it is also necessary to construct a multi-targets plasmid (MTP) molecule that can be used as the positive material. In this study, the information of the transgenic elements in 29 GM soybean events was collected and the combinations and frequencies of these elements were analyzed, to establish a novel screening protocol. It includes eight detecting targets, CaMV 35S promoter (P-35S), NOS terminator (T-nos), herbicide tolerance gene pat, E9 terminator (T-E9), insecticidal gene cry1Ac, AHAS promoter (P-AHAS), pin Ⅱ terminator (T-pin Ⅱ), and the event-specific sequence of the transgenic event DP305423, and an endogenous reference gene of soybean Lectin. After validation, the 29 GM soybean events described above can be screened by detection of the nine targets. This is referred to as the “8+1” protocol for GM soybean screening. Then these targeted sequences described in the protocol were simultaneously inserted into a cloning vector to construct the corresponding MTP pDDSC-1910. Finally, we tested whether it could be a positive plasmid. As expected, PCR analysis using pDDSC-1910 as a template showed that specific amplicons were observed with high sensitivity. Therefore, the “8+1” screening protocol for GM soybean was established, and the positive plasmid molecule pDDSC-1910 containing corresponding targets was successfully constructed. These results would facilitate the efficient screening and detection of transgenic soybeans.

Bcl-XL antisense oligodeoxynucleotide sensitizes human esophageal cancer cell line to 5-fluorouracil / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of the Bcl-XL antisense oligodeoxynucleotides (ASODN) in suppressing the Bcl-XL expression and increasing the sensitivity of esophageal cancer cell line EC9706 to 5-fluorouracil (5-Fu).</p><p><b>METHODS</b>The proliferation inhibitory rate of EC9706 was assessed by MTT, the expression of Bcl-XL was detected by RT-PCR and Western blot, and the apoptotic changes were examined by acridine orange (AO) fluorescent staining and flow cytometry, respectively.</p><p><b>RESULTS</b>In the group of ASODN combined with 5-Fu, the proliferation inhibitory rate of esophageal cancer cells was 71.58%, the expression inhibitory rate of Bcl-XL mRNA was 81.25%, the expression of Bcl-XL protein was decreased significantly. The apoptosis rates detected by AO fluorescent staining and flow cytometry were 69.5% and (63.32 +/- 9.23)%, respectively. There were significant differences as compared with the cell control group, the vacuity control group, the N-ODN group, the ASODN group and the 5-Fu group, respectively (P < 0.05).</p><p><b>CONCLUSION</b>Bcl-XL ASODN combined with 5-Fu can effectively inhibit the proliferation of esophageal cancer cells in vitro. Bcl-XL ASODN can significantly increase the sensitivity of esophageal cancer cells to 5-Fu through suppressing the expression of Bcl-XL.</p>

Inhibitory effects of transfected Bcl-XL antisense oligodeoxynucleotide on proliferation of esophageal cancer cells and growth of human esophageal carcinoma in nude mice / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the biologic effects of Bcl-XL antisense oligodeoxynucleotide (ASODN) transfected into cultured esophageal carcinoma cells and human esophageal carcinoma xenograft in nude mice.</p><p><b>METHODS</b>Cationic liposome-mediated ASODN was used to transfect esophageal carcinoma cells. RT-PCR, Western blot, MTT assay, flow cytometry and in-situ apoptosis cells detection (TUNEL detection) were used to systematically study the biological effects of the transfected cells in vitro and in vivo.</p><p><b>RESULTS</b>MTT assay showed that the proliferation of esophageal carcinoma cells in the ASODN group decreased significantly as compared with control (P < 0.05), along with a 57.3% inhibitory rate of Bcl-XL mRNA, a significant decrease of Bcl-XL protein and the apoptosis rates of (31.1 +/- 5.8)% and 35.0% by flow cytometry and TUNEL assay, respectively (P < 0.01, as compared with controls). The growth of human esophageal carcinoma in nude mice was also significantly inhibited in the ASODN group (P < 0.05), along with a significant decrease of Bcl-XL mRNA and protein expression, and also an enhanced apoptosis of the tumor cells in nude mice.</p><p><b>CONCLUSIONS</b>Bcl-XL ASODN can effectively inhibit the proliferation of esophageal carcinoma cells in vitro and the growth of the tumor in vivo. The suppression of Bcl-XL expression by ASODN may offer both a therapeutic approach and an important theoretic foundation for gene therapy against esophageal carcinoma.</p>