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Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used with many adherent and nonadherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines (the latter requires a drug selection process). This protocol is also widely used for co-expression of plasmids for packaging viruses. Efficiency of transfection can be close to 100% depending on the cell lines used. Here, a calcium phosphate transfection protocol is described using a GFP plasmid and the HEK293 cell line.

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[Bio101] Calcium Phosphate Transfection of Eukaryotic Cells
[Bio101] 磷酸钙介导的真核细胞转染方法

分子生物学 > DNA > 转染
作者: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
2/5/2012, 29390 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.86

[Abstract] Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA (Graham and van der Eb, 1973). The insoluble calcium phosphate precipitate with the attached DNA adheres to the cell surface and is brought into the cells by endocytosis. Calcium phosphate transfection has been optimized and widely used with many adherent and nonadherent cell lines (Jordan et al., 1996). Calcium phosphate transfection can result in transient expression of the delivered DNA in the target cell, or establishment of stable cell lines (the latter requires a drug selection process). This protocol is also widely used for co-expression of plasmids for packaging viruses. Efficiency of transfection can be close to 100% depending on the cell lines used. Here, a calcium phosphate transfection protocol is described using a GFP plasmid and the HEK293 cell line.

[Abstract] 小干扰RNA(siRNA)是一类20-25个核苷酸的双链RNA,在许多生物过程中起重要作用(Hamilton和Baulcombe,1999)。 siRNA通过"中和"靶蛋白的mRNA起作用,促进mRNA的降解,从而改变蛋白的生物效应(综述于Hannon和Rossi,2004)。 siRNA也可以改变调节RNA的细胞内水平。 在生物研究中使用siRNA操纵目的基因的表达通常被称为RNA干扰或敲除技术(Elbashir等人,2001)。 合成的siRNA是一种新兴的工具,现在广泛应用于这些研究。 不同公司采用多种算法来设计siRNA产品,其在功效,特异性和成本等方面不同。 在此使用来自Dharmacon的siGENOME SMARTpool试剂解释siRNA敲低的示例方案。

Materials and Reagents

  1. HEK-293 cells (ATCC, catalog number: CRL-1573 ™)
  2. Eagle's minimum essential medium (ATCC, catalog number: 30-2003 ™)
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  5. HEPES (Sigma-Aldrich, catalog number: H4034 )
  6. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  7. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
  8. pEGFP-Actin plasmid (this is an example plasmid; Clontech, catalog number: PT3265-5 )
    Note: The pEGFP-Actin Vector expresses the EGFP-Actin fusion protein in mammalian cells; this protein is incorporated into growing actin filaments and allows for visualization of actin-containing subcellular structures in living and fixed cells. http://www.clontech.com/images/pt/dis_vectors/PT3265-5.pdf
  9. 2x HBS buffer (see Recipes)
  10. Solution-A (see Recipes)
  11. Solution-B (see Recipes)

Equipment

  1. Tissue culture plates (e.g., 35 mm polystyrene)
  2. Cell culture incubator: 37 ºC and 5% CO2

Procedure

  1. Prepare 2 M CaCl2 solution in water, filter sterilize and keep at room temperature.
  2. Prepare the 2x HBS buffer (see Recipes).
  3. Carry HEK-293 cells in Eagle's Minimum Essential Medium with 10% FBS.
  4. 24 h before transfection, trypsinize and reseed log-phase cells into 35 mm tissue culture dishes. For seeding density, cells should reach 60-70% confluence for transfection.
    Note: Best confluence for different cell lines differ. For HEK-293 cells, 60-70% confluence is suggested.
  5. 3 h before transfection, replenish cells with fresh medium.
  6. For each DNA transfection, prepare mixtures in two separate tubes (Solution-A and Solution-B, see Recipes)
  7. Add Solution-B slowly (drop-wise) into Solution-A while mixing Solution-A.
    Note: This is the most important step for forming DNA/calcium phosphate co-precipitate. Mix gently but thoroughly to allow formation of precipitates evenly.
  8. After mixing the two solutions, incubate at room temperature for 20-30 min (a shorter incubation time may be used for different cell types; please determined empirically). The solution will become opaque while precipitates being formed.
  9. Gently tap the mixture. Add the mixture directly to cells by dripping slowly and evenly into medium (a good way is to let tip touch medium surface).
  10. Gently tilt the plate back-and-forth a couple of times to allow even distribution of added precipitate on the cell surface.
  11. Incubate the cells at 37 ºC with 5% CO2 for 24 h and then replenish medium.

Notes

  1. GFP expression, if used as your positive, can be detected usually after 24-48 h of cell growth.
  2. Transfection efficiency (when using HEK293 cells and the above mentioned sample plasmid) can reach up to 90-100%.

Recipes

  1. 2x HBS buffer
    50 mM   
    HEPES

    280 mM 
     NaCl

    1.5 mM   
    Na2HPO4

    Adjust pH to 7.0 using HCl. Filter sterilize. The solution can be freezed/thawed once for future use.
  2. Solution-A
    100 μl 2x HBS
  3. Solution-B
    1-5 μg DNA (e.g., pEGFP-Actin plasmid as suggested above)
    12.2 μl of 2 M CaCl2
    ddH2O to bring volume up to 100 μl, pipet gently to mix
    Note: Titration of DNA should be carried out to obtain the best efficiency of transfection.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Graham and van der Eb (1973) and Jordan et al. (1996). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee  [see Chen et al. (2009)].

References

  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. Graham, F. L. and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67.
  3. Jordan, M., Schallhorn, A. and Wurm, F. M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4): 596-601.

材料和试剂

  1. HEK-293细胞(ATCC,目录号:CRL-1573 TM)
  2. Eagle最小必需培养基(ATCC,目录号:30-2003 TM)
  3. 胎牛血清(FBS)(ATCC,目录号:30-2020 TM)
  4. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  5. HEPES(Sigma-Aldrich,目录号:H4034)
  6. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  7. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S5136)
  8. pEGFP-肌动蛋白质粒(这是实施例质粒; Clontech,目录号:PT3265-5) 注意:pEGFP-肌动蛋白载体在哺乳动物细胞中表达EGFP-肌动蛋白融合蛋白; 这种蛋白质被并入生长肌动蛋白丝,并允许在活细胞和固定细胞中含肌动蛋白的亚细胞结构的可视化。 http://www.clontech.com/images/pt/dis_vectors/PT3265-5.pdf
  9. 2x HBS缓冲区(见配方)
  10. 解决方案A(参见配方)
  11. 解决方案B(参见配方)

设备

  1. 组织培养板(例如35mm聚苯乙烯)
  2. 细胞培养孵育器:37℃和5%CO 2/h

程序

  1. 准备2 M CaCl 2水溶液,过滤灭菌并保持在室温。
  2. 准备2x HBS缓冲液(见配方)。
  3. 在含有10%FBS的Eagle's最低必需培养基中携带HEK-293细胞
  4. 在转染前24小时,胰蛋白酶消化并将对数期细胞再接种到35mm组织培养皿中。 对于播种密度,细胞应达到60-70%汇合用于转染 注意:不同细胞系的最佳融合不同。 对于HEK-293细胞,建议60-70%汇合。
  5. 转染前3小时,用新鲜培养基补充细胞
  6. 对于每个DNA转染,在两个单独的管(溶液A和溶液B,参见配方)中制备混合物
  7. 在混合溶液A时,将溶液B缓慢(逐滴)加入溶液A中 注意:这是形成DNA /磷酸钙共沉淀的最重要的步骤。 轻轻混匀,以均匀地形成沉淀。
  8. 混合两种溶液后,在室温下孵育20-30分钟(更短的温育时间可用于不同的细胞类型;请根据经验确定)。 溶液会变得不透明,同时形成沉淀
  9. 轻轻敲击混合物。 通过缓慢和均匀地滴入培养基(一种好的方法是让尖端接触介质表面)将混合物直接添加到细胞中。
  10. 轻轻地来回倾斜板几次,以使细胞表面均匀分布添加的沉淀物。
  11. 在37℃下用5%CO 2孵育细胞24小时,然后补充培养基。

笔记

  1. GFP表达,如果用作阳性,通常可以在细胞生长24-48小时后检测到
  2. 转染效率(当使用HEK293细胞和上述样品质粒时)可以达到90-100%。

食谱

  1. 2x HBS缓冲液
    50 mM   
    HEPES

    280 mM 
      NaCl

    1.5 mM   
    Na HPO 4

    使用HCl将pH调节至7.0。 过滤灭菌。 该溶液可以冷冻/解冻一次,以备将来使用
  2. 解决方案A
    100μl2x HBS
  3. 解决方案B
    1-5μgDNA(例如,如上所建议的pEGFP-肌动蛋白质粒) 12.2μl的2M CaCl 2/v/v ddH 2 O使体积达到100μl,轻轻地移液以混合
    注意:应进行DNA滴定以获得最佳的转染效率。

致谢

该方案在美国加利福尼亚州La Jolla的Scripps研究所的免疫学系中开发,并改编自Graham和van der Eb(1973)和Jordan等人(1996)。 该工作由NIH资助,CA079871和CA114059以及加利福尼亚大学的烟草相关疾病研究计划15RT-0104由Jiing-Dwan Lee博士资助[参见Chen (2009)]。

参考文献

  1. Chen,Y.,Lu,B.,Yang,Q.,Fearns,C.,Yates,J.R.,3rd和Lee,J.D。(2009)。 组合整合素磷蛋白分析和基于小干扰RNA的功能筛选确定了癌细胞粘附的关键调节因子, 。 Cancer Res 69(8):3713-3720。
  2. Graham,F.L。和van der Eb,A.J。(1973)。 用于测定人腺病毒5 DNA的感染性的新技术 Virology 52(2):456-67。
  3. Jordan,M.,Schallhorn,A。和Wurm,F.M。(1996)。 转染哺乳动物细胞:影响磷酸钙沉淀形成的关键参数的优化 Nucleic Acids Res 24(4):596-601。
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How to cite this protocol: Chen, Y. (2012). Calcium Phosphate Transfection of Eukaryotic Cells. Bio-protocol Bio101: e86. DOI: 10.21769/BioProtoc.86; Full Text



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7/21/2014 11:14:29 AM  

Hailong Zhang
Department of Pediatrics, Emory University, USA

Hi, Thank you for your protocol, I have a question: in step "9" you mix the two solutions at room temperature for 20-30 minutes. but I see some other protocols including your references saying 1 minute is enough. prolonged incubation would decrease the DNA binding efficient.
What's your suggestion? please confirm.
Thanks a lot.

7/25/2014 8:43:11 AM  

Yanling Chen (Author)
Department of Immunology, The Scripps Research Institute, USA

The incubation time needs to be determined empirically for different cell types. Yes, a shorter incubation time can be used. Thanks.

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10/17/2012 9:38:23 AM  

Thanks a lot

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