miR-223敲减慢病毒的构建

目的构建mi R-223敲减慢病毒,为进一步研究mi R-223的功能提供工具。方法采用Invitrogen公司mi R-RNAi在线设计工具,根据mi R-223成熟体序列设计了一对互补寡核苷酸片段,退火产物连接至pc DNA6.2-GW/Em GFP-mi R载体,经酶切连入p CDH-CMV-MCS-EF1-cop GFP载体,构建了mi R-223敲减慢病毒质粒,利用293T细胞将其包装成病毒,并感染ST2细胞,观察感染效率并用q RT-PCR检测mi R-223成熟体表达。结果酶切鉴定及测序结果显示成功构建了mi R-223敲减慢病毒载体,mi R-223敲减慢病毒包装并感染靶细胞,...

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Bibliographic Details
Published in天津医药 Vol. 43; no. 7; pp. 717 - 720
Main Author 张欣 关晓慧 王宝利
Format Journal Article
LanguageChinese
Published 天津医科大学代谢病医院内分泌研究所 卫生部激素与发育重点实验室 邮编300070 2015
Subjects
miR-223
RNA干扰
微RNAs
慢病毒属
慢病毒源性载体
RNA干扰
miR-223
microRNAs
慢病毒属
RNA interference
lentivirus based vector
慢病毒源性载体
微RNAs
lentivirus
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ISSN0253-9896
DOI10.11958/j.issn.0253-9896.2015.07.004

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Summary:目的构建mi R-223敲减慢病毒,为进一步研究mi R-223的功能提供工具。方法采用Invitrogen公司mi R-RNAi在线设计工具,根据mi R-223成熟体序列设计了一对互补寡核苷酸片段,退火产物连接至pc DNA6.2-GW/Em GFP-mi R载体,经酶切连入p CDH-CMV-MCS-EF1-cop GFP载体,构建了mi R-223敲减慢病毒质粒,利用293T细胞将其包装成病毒,并感染ST2细胞,观察感染效率并用q RT-PCR检测mi R-223成熟体表达。结果酶切鉴定及测序结果显示成功构建了mi R-223敲减慢病毒载体,mi R-223敲减慢病毒包装并感染靶细胞,表达绿色GFP荧光蛋白的细胞约占细胞总数的80%-90%,病毒滴度为1×10^9PFU/m L,感染效率达90%。感染mi R-223敲减慢病毒的细胞mi R-223表达量（0.31±0.03）低于阴性对照（1.00±0.09）,为阴性对照的31%,差异有统计学意义（n=3,t=15.091,P〈0.05）。结论成功构建了mi R-223敲减慢病毒,为进一步研究mi R-223的功能准备了条件。
Bibliography:ZHANG Xin, GUAN Xiaohui, WANG Baoli（ Key Laboratory of Hormones and Development （Ministry of Health）, Metabolic Diseases Hospital ＆ Institute of Endocrinology, Tianjin Medical University, Tianfin 300070, China）
micro RNAs; lentivirus; RNA interference; lentivirus based vector; mi R-223
12-1116/R
Objective To construct mi R-223 knockdown lentivirus vector and provide a tool for further study of thefunction of mi R-223. Methods According to the Invitrogen mi R-RNAi online design tool, a pair of complementary oligo-nucleotides encoding mi R-223 mature sequence was designed, annealed and ligated with pc DNA6.2-GW/Em GFP-mi R vec-tor. Then mi R-RNAi expression cassette was cut and subcloned into lentiviral p CDH-CMV-MCS-EF1-cop GFP vector. Thelentiviruses were packaged and titered, and then ST2 cells were infected with viruses. The efficiency of infection was calcu-lated, and the knockdown of endogenous mi R-223 was detected by using real-time RT-PCR. Results Restriction enzymedigestion and sequencing results showed
ISSN:0253-9896
DOI:10.11958/j.issn.0253-9896.2015.07.004