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Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

Orlova NA, Kovnir SV, Hodak JA, Vorobiev II, Gabibov AG, Skryabin KG - BMC Biotechnol. (2014)

Bottom Line: Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other.The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1.The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Mammalian Cell Bioengineering, Centre "Bioengineering", Russian Academy of Sciences, 60-letija Oktyabrya 7, Moscow 117312, Russia. ptichman@gmail.com.

ABSTRACT

Background: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.

Results: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.

Conclusions: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

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eGFP-expressing cell colonies obtained by elevated selection pressure and by targeted gene amplification. Red bars: mean eGFP levels for sets of cell colonies analysed. Concentrations represent final concentrations of MTX used. A. eGFP levels for 10 colonies obtained in the absence of MTX and in the presence of 50 nM MTX, colonies were obtained by the direct plating of transiently transfected cells. B. eGFP levels for 8 colonies obtained by growth in the presence of various MTX concentrations. Polyclonal stably transfected cell population was used for plating. C. Level of intracellular eGFP in polyclonal cell populations, obtained by primary selection in presence of 50 nM MTX and subsequent amplification in presence of various concentrations of MTX. Error bars indicate the standard deviation, n = 2. D. Number of copies of genome-integrated plasmids measured by Q-PCR for populations from panel C. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3.
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Figure 4: eGFP-expressing cell colonies obtained by elevated selection pressure and by targeted gene amplification. Red bars: mean eGFP levels for sets of cell colonies analysed. Concentrations represent final concentrations of MTX used. A. eGFP levels for 10 colonies obtained in the absence of MTX and in the presence of 50 nM MTX, colonies were obtained by the direct plating of transiently transfected cells. B. eGFP levels for 8 colonies obtained by growth in the presence of various MTX concentrations. Polyclonal stably transfected cell population was used for plating. C. Level of intracellular eGFP in polyclonal cell populations, obtained by primary selection in presence of 50 nM MTX and subsequent amplification in presence of various concentrations of MTX. Error bars indicate the standard deviation, n = 2. D. Number of copies of genome-integrated plasmids measured by Q-PCR for populations from panel C. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3.

Mentions: At the same time, stable integration rate (or rate of establishment of stable episomal maintenance) of the p1.1eGFP plasmid was 24 times higher than that of the p1.1(EBVTR-)eGFP control plasmid in the selection medium lacking both HT and MTX (Table 2), clearly indicating that the EBVTR element was active in the very large expression plasmid. Transfection and selection of stably transformed CHO DG44 cells by the p1.1eGFP plasmid was repeated with the selection medium supplemented with 50 nM MTX. In this case, the eGFP expression level increased twice for the ten most productive wells (Figure 4A). Thus, the p1.1 plasmid is suitable for creation of stably transfected cell clones or populations under variable selection stringencies.


Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

Orlova NA, Kovnir SV, Hodak JA, Vorobiev II, Gabibov AG, Skryabin KG - BMC Biotechnol. (2014)

eGFP-expressing cell colonies obtained by elevated selection pressure and by targeted gene amplification. Red bars: mean eGFP levels for sets of cell colonies analysed. Concentrations represent final concentrations of MTX used. A. eGFP levels for 10 colonies obtained in the absence of MTX and in the presence of 50 nM MTX, colonies were obtained by the direct plating of transiently transfected cells. B. eGFP levels for 8 colonies obtained by growth in the presence of various MTX concentrations. Polyclonal stably transfected cell population was used for plating. C. Level of intracellular eGFP in polyclonal cell populations, obtained by primary selection in presence of 50 nM MTX and subsequent amplification in presence of various concentrations of MTX. Error bars indicate the standard deviation, n = 2. D. Number of copies of genome-integrated plasmids measured by Q-PCR for populations from panel C. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4067061&req=5

Figure 4: eGFP-expressing cell colonies obtained by elevated selection pressure and by targeted gene amplification. Red bars: mean eGFP levels for sets of cell colonies analysed. Concentrations represent final concentrations of MTX used. A. eGFP levels for 10 colonies obtained in the absence of MTX and in the presence of 50 nM MTX, colonies were obtained by the direct plating of transiently transfected cells. B. eGFP levels for 8 colonies obtained by growth in the presence of various MTX concentrations. Polyclonal stably transfected cell population was used for plating. C. Level of intracellular eGFP in polyclonal cell populations, obtained by primary selection in presence of 50 nM MTX and subsequent amplification in presence of various concentrations of MTX. Error bars indicate the standard deviation, n = 2. D. Number of copies of genome-integrated plasmids measured by Q-PCR for populations from panel C. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3.
Mentions: At the same time, stable integration rate (or rate of establishment of stable episomal maintenance) of the p1.1eGFP plasmid was 24 times higher than that of the p1.1(EBVTR-)eGFP control plasmid in the selection medium lacking both HT and MTX (Table 2), clearly indicating that the EBVTR element was active in the very large expression plasmid. Transfection and selection of stably transformed CHO DG44 cells by the p1.1eGFP plasmid was repeated with the selection medium supplemented with 50 nM MTX. In this case, the eGFP expression level increased twice for the ten most productive wells (Figure 4A). Thus, the p1.1 plasmid is suitable for creation of stably transfected cell clones or populations under variable selection stringencies.

Bottom Line: Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other.The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1.The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Mammalian Cell Bioengineering, Centre "Bioengineering", Russian Academy of Sciences, 60-letija Oktyabrya 7, Moscow 117312, Russia. ptichman@gmail.com.

ABSTRACT

Background: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure.

Results: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative.

Conclusions: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

Show MeSH
Related in: MedlinePlus