[1]罗静,蒋文,刘林,等.RPS14基因重组慢病毒载体的构建及其在MUTZ-8细胞中的表达[J].第三军医大学学报,2011,33(21):2252-2255.
 Luo Jing,Jiang Wen,Liu Lin,et al.Construction of RPS14 recombinant lentiviral vector and its expression in MUTZ-8 cells[J].J Third Mil Med Univ,2011,33(21):2252-2255.
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RPS14基因重组慢病毒载体的构建及其在MUTZ-8细胞中的表达(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第21期
页码:
2252-2255
栏目:
论著
出版日期:
2011-11-15

文章信息/Info

Title:
Construction of RPS14 recombinant lentiviral vector and its expression in MUTZ-8 cells
作者:
罗静蒋文刘林王利
重庆医科大学附属第一医院血液科
Author(s):
Luo JingJiang Wen Liu Lin Wang Li
Department of Hematology, First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China
关键词:
RPS14基因慢病毒载体骨髓增生异常综合征
Keywords:
RPS14 lentiviral vector myelodysplastic syndrome
分类号:
R394-33; R394.3; R552.02
文献标志码:
A
摘要:
目的    构建携带人RPS14基因重组慢病毒载体获得稳定产毒的细胞,并观察其在人MDS细胞株MUTZ-8细胞中的表达。    方法      通过反转录方法,获得目的基因RPS14;将目的基因克隆入慢病毒表达质粒pGC-FU Vector,构建含有RPS14基因的重组慢病毒载体pGC-FU RPS14,经PCR、酶切及测序鉴定其正确性;在Lipofectamine 2000介导下,将成功构建的质粒pGC-FU RPS14与慢病毒包装质粒pHelper 1.0、pHelper 2.0共转染293T细胞,通过实时定量PCR法测定病毒滴度,并将获得的重组慢病毒载体pGC-FU RPS14转染人MDS细胞株MUTZ-8,用流式细胞仪检测转染效率,RT-PCR检测靶细胞内RPS14 mRNA的表达。    结果      含有RPS14基因的重组慢病毒载体经PCR、酶切和测序鉴定构建正确,且测定的病毒滴度为2.0×109 TU/ml。流式细胞仪检测其转染效率为68.41%,RT-PCR检测到转染后RPS14 mRNA在靶细胞中的稳定表达。    结论      构建了携带人RPS14 基因的慢病毒载体,转染人MUTZ-8细胞株后能够稳定表达RPS14基因。
Abstract:
Objective      To construct a recombinant lentiviral vector carrying RPS14 gene, establish a stable packaging 293T cell lines, and observe RPS14 expression in human MDS cell line MUTZ-8.     Methods      RPS14 was obtained using reverse transcription polymerase chain reaction (RT-PCR). Then the RPS14 gene was cloned into a lentiviral vector pGC-FU to construct a recombinant lentiviral vector carrying RPS14 gene named pGC-FU RPS14, which was confirmed by PCR, restriction enzyme digestion and DNA sequencing. With the help of Lipofectamine 2000, 293T cells were cotransfected with pGC-FU RPS14, pHelper 1.0 and pHelper 2.0. The titer of the virus was examined by real-time PCR. pGC-FU RPS14 was used to transfect human MDS cell line MUTZ-8. The transfection efficiency and RPS14 mRNA expression were detected by flow cytometry and RT-PCR, respectively.     Results      The recombinant lentiviral vector pGC-FU RPS14 was confirmed to be correctly constructed by PCR, restriction enzyme digestion and DNA sequencing; the assayed titer of virus was 2.0×109 Tu/ml. The transfection efficiency was 68.41%, and stable expression of RPS14 mRNA in MUTZ-8 cells was detected by RT-PCR.     Conclusion      A lentiviral vector carrying RPS14 gene is successfully constructed, which can deliver RPS14 gene into human MDS cell line MUTZ-8 for RPS14 stable expression.

参考文献/References:

罗静, 蒋文, 刘林, 等. RPS14基因重组慢病毒载体的构建及其在MUTZ-8细胞中的表达[J].第三军医大学学报,2011,33(21):2252-2255.

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更新日期/Last Update: 2011-11-08