Optimizing conditions for calcium phosphate mediated transient transfection

Ling Guo, Liyang Wang, Ronghua Yang, Rui Feng, Zhongguang Li, Xin Zhou, Zhilong Dong, George Ghartey-Kwansah, Meng Meng Xu, Miyuki Nishi, Qi Zhang, William B Isaacs, Jianjie Ma, Xuehong Xu

Research output: Contribution to journalArticle

Abstract

Background Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. Results In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate–DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells. Conclusions Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

Original languageEnglish (US)
Pages (from-to)622-629
Number of pages8
JournalSaudi Journal of Biological Sciences
Volume24
Issue number3
DOIs
StatePublished - Mar 1 2017

Fingerprint

calcium phosphates
transfection
Transfection
Shock
Glycerol
glycerol
recombinant DNA
Recombinant DNA
cultured cells
cells
calcium phosphate
Cultured Cells
DNA
Buffers
cell culture
buffers
Cell Culture Techniques
HEPES
coprecipitation
Costs and Cost Analysis

Keywords

  • Calcium phosphate transfection
  • Co-precipitation
  • Transfection efficiency

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

Optimizing conditions for calcium phosphate mediated transient transfection. / Guo, Ling; Wang, Liyang; Yang, Ronghua; Feng, Rui; Li, Zhongguang; Zhou, Xin; Dong, Zhilong; Ghartey-Kwansah, George; Xu, Meng Meng; Nishi, Miyuki; Zhang, Qi; Isaacs, William B; Ma, Jianjie; Xu, Xuehong.

In: Saudi Journal of Biological Sciences, Vol. 24, No. 3, 01.03.2017, p. 622-629.

Research output: Contribution to journalArticle

Guo, L, Wang, L, Yang, R, Feng, R, Li, Z, Zhou, X, Dong, Z, Ghartey-Kwansah, G, Xu, MM, Nishi, M, Zhang, Q, Isaacs, WB, Ma, J & Xu, X 2017, 'Optimizing conditions for calcium phosphate mediated transient transfection', Saudi Journal of Biological Sciences, vol. 24, no. 3, pp. 622-629. https://doi.org/10.1016/j.sjbs.2017.01.034
Guo, Ling ; Wang, Liyang ; Yang, Ronghua ; Feng, Rui ; Li, Zhongguang ; Zhou, Xin ; Dong, Zhilong ; Ghartey-Kwansah, George ; Xu, Meng Meng ; Nishi, Miyuki ; Zhang, Qi ; Isaacs, William B ; Ma, Jianjie ; Xu, Xuehong. / Optimizing conditions for calcium phosphate mediated transient transfection. In: Saudi Journal of Biological Sciences. 2017 ; Vol. 24, No. 3. pp. 622-629.
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abstract = "Background Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. Results In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate–DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells. Conclusions Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.",
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T1 - Optimizing conditions for calcium phosphate mediated transient transfection

AU - Guo, Ling

AU - Wang, Liyang

AU - Yang, Ronghua

AU - Feng, Rui

AU - Li, Zhongguang

AU - Zhou, Xin

AU - Dong, Zhilong

AU - Ghartey-Kwansah, George

AU - Xu, Meng Meng

AU - Nishi, Miyuki

AU - Zhang, Qi

AU - Isaacs, William B

AU - Ma, Jianjie

AU - Xu, Xuehong

PY - 2017/3/1

Y1 - 2017/3/1

N2 - Background Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. Results In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate–DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells. Conclusions Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

AB - Background Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research. Results In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate–DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells. Conclusions Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.

KW - Calcium phosphate transfection

KW - Co-precipitation

KW - Transfection efficiency

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