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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, 27131 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] 用DNA转染细胞是分子生物学中一个不可缺少的方法。虽然脂转染试剂已实现商业化,此外还有一项十分快速,简便,高效,廉价,既通过磷酸钙与DNA的共沉淀转染真核细胞的方法。(1)DNA附着在不可溶的磷酸钙沉淀上,粘合到细胞表面后,共同通过内吞作用被带入细胞内部。经过优化的磷酸钙转染被广泛应用用许多粘合或不粘合的细胞系中。(2)DNA进入靶细胞,可以是瞬时的也可以建立稳定的细胞系,这个方法被广泛用于组装病毒。就不同的细胞株转染效率可以接近100%。这里GFP的质粒和HEK293细胞为例,介绍利用CaPO4转染的具体方法。

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,试剂和设备

 

1.       HEK-293细胞(ATCC#的CRL- 1573

2.       鹰的最低基本培养基ATCC30-2003

3.       胎牛血清(ATCC30-2020

4.       氯化钙CaCl2 (Sigma - Aldrich公司#C5670

5.       HEPESSigma - Aldrich公司#H4034

6.       氯化钠NaClSigma - Aldrich公司#S5886

7.       磷酸氢二钠Na2HPO4Sigma - Aldrich公司#S5136

8.       组织培养板(例如,35毫米聚苯乙烯板)

9.       细胞培养孵化器:375%二氧化碳

10.   载体pEGFP-Actin质粒(这是一个例子质粒; Clontech公司#PT3265- 5

公司注释: pEGFP-Actin肌动蛋白表达载体,能够在哺乳动物细胞中表达肌动蛋白和绿色荧光蛋白GFP的融合蛋白,这种蛋白微丝参与到生长肌动蛋白片段,使含有机动蛋白的活细胞的亚细胞结构可视化。 http://www.clontech.com/images/pt/dis_vectors/PT3265-5.pdf

 

步骤:

 

1.       准备2M CaCl2 抽滤后室温放置

2.       准备2x HBS buffer  含有

50 mM             HEPES

280 mM                  NaCl

1.5 mM                        Na2HPO4

HClPH7.0抽滤消毒。该溶液可以冻融一次以供将来使用。

3.       HEK- 293细胞放置在含有10%胎牛血清的鹰最低基本培养基中。

4.       转染24小时,将胰蛋白酶消化的对数期细胞转接入35毫米组织培养板 中。接种confluence应,细胞达到转染所需confluence60?70%。

注:不同的细胞系的最佳 confluence不同。对于染HEK- 293细胞建议达到60-70%。

5.       转染前3小时,补充新鲜培养基。

6.       对于每个DNA转染,在两个独立的管中准备以下混合物:

溶液A100微升2x HBS

溶液B1-5微克的DNA(如pEGFP-肌动蛋白质粒)

12.2微升2M CaCl2

双蒸水,定容至100微升,吸管轻柔混合。

注:对DNA进行滴定,可以取得最佳的转染效率。

7.       将溶液B缓慢滴入溶液A

8.       注意:该步骤是形成DNA /磷酸钙沉淀的关键步骤。轻轻的彻底混匀,形成均匀沉淀。

9.       混合物置于室温孵育20-30分钟,沉淀的形成使混合液呈不透明。

10.   轻轻敲打的混合物。直接将混合物慢慢地,均匀地滴入到细胞中(推荐让枪头尖接触介质表面)。

11.   轻轻来回颠倒几次,以便沉淀均匀地吸附在细胞表面。

12.   375CO2培养箱中孵育24小时,补充培养基。

注:a GFP的表达在细胞生长后24-48小时进行观察。

b效率可以达到90-100%。

 

引用文献

 

1.        Graham F.L., van der Eb A.J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52(2): 456-67. 

2.        Jordan M., Schallhorn A., Wurm F.M. (1996). Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. 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
Emory University

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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