构建骨肉瘤OS-9607细胞cDNA文库及其质量分析(英文)

Construction of cDNA library of osteosarcoma cell OS-9607 and its quality analysis

背景:骨肉瘤OS-9607细胞是一种从人骨肉瘤组织中培养出的细胞株。构建该细胞系cDNA文库,以便从中克隆目的基因。目的:通过SMART技术(一种以RNA转录时5’末端转换机制为原理的技术)构建骨肉瘤OS-9607细胞cDNA文库并进行质量分析。设计:验证性的实验研究。单位:解放军第二军医大学长海医院骨科。材料:实验于2005-05在解放军第二军医大学长海医院骨科完成。骨肉瘤OS-9607细胞在CO2培养箱中培养。细胞先用DM处理30min,然后在标准培养基中恢复24h。骨肉瘤OS-9607细胞的全部RNA可通过RNAease试剂盒分离。全部RNA的数量及完整性通过紫外线光谱分析器和溴乙锭染色的琼脂糖凝胶电泳检测。方法:从人骨肉瘤OS-9607细胞中提取全部RNA并分离出mRNA,通过原位聚合酶链反应和长距离聚合酶链反应技术获得OS-9607细胞的cDNA,最后将cDNA与去磷酸化的λ噬菌体连接。重组物是用SMARTcDNA文库试剂盒构建的。插入的cDNA长短和文库的多样性通过聚合酶链反应检测。主要观察指标:总RNA的数量和完整性,未扩增文库和扩增文库的滴度及重组指数,分析文库中cDNA长度。结果:①总RNA的浓度测得其吸光谱位于260～280nm,其吸光度大约为1.9A,可清晰的显示其为28SrRNA和18SrRNA,其亮度比值为2∶1(28S/18S)。②在试管内与λ噬菌体连接并转染宿主细菌后,未扩增文库的滴定度达到2.2×107pfu/mL,而扩增文库达到4.5×1010pfu/mL;重组指数分析,在一个培养基中蓝色斑点有8个,无色斑点达到160个,重组指数为99.5%。③获得的cDNA文库由1.6×106个重组噬菌体组成,重组物中平均外源插入物达到1.5kb。结论:本研究获得的骨肉瘤OS-9607细胞cDNA文库质量优良,为今后骨肉瘤OS-9607细胞方面的相关研究及骨肉瘤相关基因的筛选奠定了坚实的基础。

BACKGROUND： Osteosarcoma cell OS-9607 is a cell line cultured from human osteosarcoma tissue. To construct cDNA library of this cell line so as to clone target gene from it. OBJECTIVE： To construct cDNA library of osteosarcoma cell 0S-9607 by switching mechanism at 5＇ end of RNA transcript （SMART） technique and analyze its quality. DESIGN： Verified experimental study SETTING： Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA MATERIALS： This experiment was carried out at the Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA in May 2005. Osteosarcoma cell OS-9607 was cultured in CO2 incubator. Cells were pre-treated with DM for 30 minutes, recovered for 24 hours in standard culture medium. The whole RNA of osteosarcoma cell OS-9607 was isolated by RNAease reagent kit. The quantity and integrity of total RNA was detected by ultraviolet spectrometer and electrophoresis on a denaturing formaldehyde agarose gel stained by ethidium bromide （EtBr）. METHODS： Total RNA was extracted from human osteosarcoma cell OS- 9607 and mRNA was isolated, cDNA of OS-9607 cells was acquired by in situ polymerase chain reaction （PCR） and long-distance PCR （LD-PCR）,then the cDNA was ligated with dephosphorylated h phage vector. The recombinants were constructed with SMART cDNA library construction kit. The size of cDNA inserts and the diversity of library were detected by PCR. MAIN OUTCOME MEASURES： The quality and integrity of total RNA, the titer of un-amplified and amplified library and recombination fraction; analysis of cDNA length of library RESULTS： （1）The concentration of the isolated total RNA was determined and the measuring absorbance was between 260 nm and 280 nn. Its absorptance was about 1.9 A, 0.8% denaturing formaldehyde agarose gel electrophorosis showed clear 28S rRNA and 18S rRNA, and the brightness rate of 28S rRNA and 18S rRNA （28S/18S） was 2：1. （2）After linked to λ phage in vitro, packed and transfected host bacteria, the titer of unamplified library was 2,2×10^7 pfu/mL, and the titer of amplified library arrived to 4.5×10^10 pfu/mL. The recombination fraction analysis showed that there were 8 blue plaques and 1 360 colorless plaques in one plate, so the recombination fraction was 99.5%, （3） The obtained cDNA library consisted of 1.6×10^6 recombinant bacteriophages and the average exogenous inserts of the recombinants was 1.5 kb. CONCLUSION： These results suggest that the obtained osteosarcoma cell OS-9607 cDNA library has high quality, which sets up a finn foundation for the further research on osteosarcoma cell OS-9607 and its related genes screen