Search | Engineering

订阅投稿

首页工程期刊工程焦点工程成就工程前沿关于我们 English

资源类型

期刊论文 3

年份

2019 1

2009 2

关键词

检索范围：

排序：展示方式：

Gene silencing efficiency of shRNA expression vectors targeting Cx43

Cuihong ZHENG, Yunxia WU, Guangying HUANG, Wei WANG

《医学前沿（英文）》 2009年第3卷第2期页码 130-135 doi: 10.1007/s11684-009-0030-9

摘要： Our previous studies showed that there were close relationships between connexin 43 (Cx43) and acupoints and meridians. In order to further investigate the effect of Cx43 in acupuncture treatment, RNA interference technique was used to construct small hairpin RNA (shRNA) expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for further use . Aiming directly at the two targets of Cx43 mRNA sequence of the rat and mouse homology region, we synthesized two pairs of complementary oligonucleotide strands . Double strands were formed after annealing, and then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombinant plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into the NIH/3T3 cells. Immunofluorescence and Western blot assays were used to detect the protein level of Cx43 after being screened by G418.The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43, and a control expression vector for rat and mouse. Also, the Cx43 protein level was decreased by 73.5% ( < 0.01) and 10.8%, accordingly. The Cx43 protein level was not influenced by the transfection of P-con-shRNA. The outcomes demonstrate that the plasmid P-Cx43-shRNA (1) can specifically silence better the expression of Cx43 in NIH/3T3 cells, which offers an experimental evidence for further investigation.

关键词： RNA interference connexin 43 small hairpin RNA (shRNA) acupuncture

HTML PDF 收藏

Construction of multiple shRNA vectors targeting PEDV and TGEV and production of transgenic SCNT porcine

Jianwen CHEN, Kaiyuan PAN, Zhen CHEN, Biao DING, Dandan SONG, Wenbin BAO, Yunhai ZHANG

《农业科学与工程前沿（英文）》 2019年第6卷第1期页码 66-72 doi: 10.15302/J-FASE-2018229

摘要：

Porcine viral diarrhea is an acute and highly contagious enteric disease of pigs that causes huge economic losses worldwide. Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are the main pathogens responsible for piglet viral diarrhea. However, currently there is no specific drug available for the effective treatment of viral diarrhea. Therefore, it is necessary to seek an effective method to diminish PEDV and TGEV infection rates. RNA interference has been applied successfully to inhibit the virus replication. It provides a potential strategy for breeding resistant pigs. In this study, four promoters and four short hairpin RNA (shRNA) vectors with LoxP sites at each end of the selectable marker genes were constructed to target PEDV and TGEV. These vectors were then transfected into porcine fetal fibroblasts, G418 resistant transfectants were confirmed by PCR and transgenic SCNT porcine blastocysts were obtained. These results have paved the way for future production of marker-free transgenic resistant to PEDV and TEGV pigs by SCNT.

关键词： piglet diarrhea RNAi PEDV TGEV transgenic resistance breeding

HTML PDF 收藏

Effect of pEGFP-survivin on GBC-SD cell growth and chemotherapy sensitivity

Hanbin SHEN MD, Bin ZHANG MBA, Song ZHAO BM, Qichang ZHENG MD, Jianping GONG MD, Xiaotang CAI BM,

《医学前沿（英文）》 2009年第3卷第4期页码 485-490 doi: 10.1007/s11684-009-0089-3

摘要： This paper is aimed to investigate the effect of survivin shRNA on chemotherapy resistance in human gallbladder carcinoma GBC-SD cells. The viability of human gallbladder carcinoma GBC-SD, GBC-SD/enhanced green fluorescent protein (EGFP), and GBC-SD/survivin cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and mRNA and protein of survivin were tested by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After the cells were treated with cisplatin (DDP) (3.0 μg/mL) for the same time, cell survival rate and IC was detected with MTT, cell apoptosis was detected with fluorescence-activated cell sorting (FACS), and the nuclear alteration was observed by TdT-mediated deoxyuridine triphosphate nick end labeling (TUNEL). In addition, caspase-3 activity was detected by using colorimetric method. Cell viability was decreased significantly in GBC-SD/survivin cells, and survivin expression was decreased significantly (mRNA and protein of survivin were decreased by 74.7% and 71.5%, respectively). After treatment with DDP, cell survival rate and IC was decreased significantly (2.03±0.24 μg/mL) in GBC-SD/survivin cells, while apoptotic rate (84.3%) was elevated significantly as compared with the other two groups. There were brown apoptotic nuclei in all the cells. Caspase-3 activity in all the cells was increased at first and then decreased, but the caspase-3 activity in GBC-SD/survivin cells was significantly higher than the other two groups. The survivin shRNA could down-regulate the expression of survivin in GBC-SD cells significantly and improve the sensibility to chemotherapy.

关键词： survivin shRNA chemotherapy sensibility GBC-SD