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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2015, Vol. 41 Issue (4): 407-413    DOI: 10.3785/j.issn.1008-9209.2014.12.081
生物科学与技术     
一种高效稳定的磷酸钙转染HEK293T细胞的方法
况野，房有荣，刘丽，王蓉，严子琴，李海龙，孟凡国，盛清，欧文斌
1.浙江理工大学生命科学学院，杭州 310018；2.浙江清华长三角研究院，浙江省应用酶学重点实验室，浙江 嘉兴 314006
A high-efficiency and stable transfection approach by using calcium phosphate in HEK293T cells
Kuang Ye, Fang Yourong, Liu Li, Wang Rong, Yan Ziqin, Li Hailong, Meng Fanguo, Sheng Qing, Ou Wenbin
(1. College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China; 2. Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, Zhejiang, China)
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摘要： 为了克服磷酸钙转染法在真核细胞中转染效率低和不稳定性等缺点，适用于体外在哺乳细胞中高效表达外源蛋白，将绿色荧光蛋白（green fluorescent protein，GFP）表达质粒通过常规的磷酸钙转染法导入到HEK293T细胞中，对影响质粒转染效率的条件逐步予以优化，具体包括：在制备磷酸钙-DNA沉淀复合物时，对DNA质粒的用量和涡旋混匀时间的优化；磷酸钙-DNA沉淀形成后对转染促进剂（甘油、丁酸钠和氯喹）的浓度、作用时间及其组合进行优化和筛选.结果表明：GFP转染6孔板培养的HEK293T细胞的DNA质粒最适用量为3 μg/孔，其转染效率较1和2 μg/孔的平均转染效率提高了50%，较4和5 μg/孔的平均转染效率提高了70%以上；涡旋混匀时间最佳为10 s，比涡旋混匀5 s时的转染效率提高了80%；在上述优化条件基础上加入不同的转染促进剂，其中单独使用15%甘油处理细胞6.5 min时，所得转染效率较2.5、4.5、8.5和10.5 min有明显提高，15%甘油+5 mmol/L丁酸钠联用或者单独用500 μmol/L氯喹处理细胞后，转染效率较常规方法分别提高了80%和75%，且荧光表达强度有所增强.总之，该文通过对磷酸钙转染时和转染后的条件进行优化，使其转染效率较常规方法有了显著提高，相对于脂质体转染法更加安全可靠，且价格低廉，因此可适用于大规模蛋白表达质粒转染或者病毒包装，尤其适用于各种糖基化蛋白如肿瘤诊断标志物在体外的高通量表达与制备.
Abstract: Transfection, involving the transfer of exogenous DNA to eukaryocyte cells, is a common technique in molecular and cellular biology, including calcium phosphate transfection, electroporation, lipofection and so on. Calcium phosphate transfection is one of the major methods for DNA transfer to eukaryocyte cells and with lower toxicity and cost than lipofection. However, the application of calcium phosphate transfection method was limited due to its low transfection efficiency and instability. 
To establish a highly efficient and stable transfection approach to overexpress exogenous protein abundantly in HEK293T cells in vitro, green fluorescent protein (GFP) plasmid DNA was transfected by using calcium phosphate with optimal DNA amount and vortex time of calcium phosphateDNA complex in absence or presence of common transfection enhancers including glycerol, sodium butyrate, and chloroquine. 
The protocol of calcium phosphate transfection was as follows. First, HEK293T cells were seeded in a sixwell plate, incubated for 8-24 hours at 37 ℃ in a humid incubator with 5% CO2, and then the cells were transfected after cell confluence reached 50%-80%. Second, calcium phosphate-DNA complex was prepared. Mixed GFP DNA plasmid with 25 μL of 2.5 mol/L CaCl2 solution in a roundbottom tube, added sterile water up to 250 μL, blended 10 μL of phosphatesolution ［V(70 mmol/L Na2HPO4)∶〖V(70 mmol/L NaH2PO4)=1∶1］ with 250 μL of 2×Hepes buffer solution and added to the former solution, added calcium phosphateDNA mixture into HEK293T cells one drop at a time immediately after vortex. Finally, transfection efficiency was detected by fluorescence imaging in absence or presence of common transfection enhancers including glycerol, sodium butyrate, and chloroquine. The dosage of DNA, duration of vortex and concentrations of three common transfection enhancers were optimized during or after transfection. 
The results showed that the optimal GFP plasmid DNA amount was 3 μg/well. Compared with GFP plasmid DNA amount of 1 or 2 μg/well and 4 or 5 μg/well, average transfection efficiency of optimal amount was improved by 50% and 70%, respectively. Higher transfection efficiency was also detected when the vortex time was 10 s. Under these optimal conditions, transfection efficiency of treatment with 15% glycerol alone for 6.5 min was significantly higher than that for 2.5, 4.5, 8.5, or 10.5 min. When the HEK293T cells were treated with the combination of 15% glycerol with 5 mmol/L sodium butyrate or 500 μmol/L chloroquine alone, transfection efficiency was improved by 80% and 75%, respectively. Meanwhile, GFP fluorescence intensity was enhanced. 
Compared to lipofectamine reagents, calcium phosphate transfection with enhancers is more efficient, less toxic and less expensive. Therefore, this approach can be used to overexpress largescale proteins in the HEK293T cells in vitro, or to package lentiviruses/retroviruses. It is especially applicable for in vitro high flux expression and preparation for various glycosylated proteins such as cancer diagnosis markers.
出版日期: 2015-07-20
CLC:  Q 2  
通讯作者: 盛清（http://orcid.org/0000-0003-1326-8674）;欧文斌（http://orcid.org/0000-0002-6311-9442）     E-mail: csheng866@126.com;ouwenbin@tsinghua.org.cn
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况野
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引用本文:

况野，房有荣，刘丽，王蓉，严子琴，李海龙，孟凡国，盛清，欧文斌. 一种高效稳定的磷酸钙转染HEK293T细胞的方法[J]. 浙江大学学报(农业与生命科学版), 2015, 41(4): 407-413.

Kuang Ye, Fang Yourong, Liu Li, Wang Rong, Yan Ziqin, Li Hailong, Meng Fanguo, Sheng Qing, Ou Wenbin. A high-efficiency and stable transfection approach by using calcium phosphate in HEK293T cells. Journal of Zhejiang University (Agriculture and Life Sciences), 2015, 41(4): 407-413.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2014.12.081    或    http://www.zjujournals.com/agr/CN/Y2015/V41/I4/407

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