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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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Properties of the cell populations stably transfected by p1.2-based plasmids under various drug selection stringencies. DG44: untransfected CHO DG44 cells; p1.1: cells stably transfected by the p1.1eGFP plasmid and selected in the presence of 200 nM MTX; p1.1(EBVTR-): transfection by the p1.1(EBVTR-)eGFP plasmid using the same conditions. A. Level of intracellular eGFP in cell populations. Error bars indicate the standard deviation, n = 2. B. Proportion of eGFP-negative cell populations measured by FACS. C. Number of copies of genome-integrated plasmids measured by Q-PCR. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3-4. The apparent level of the eGFP ORF DNA for the untransfected CHO DG44 cells is below 0.1 copies per one haploid genome. D. Codes for the different cell populations and the concentrations of antibiotics employed.
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Figure 3: Properties of the cell populations stably transfected by p1.2-based plasmids under various drug selection stringencies. DG44: untransfected CHO DG44 cells; p1.1: cells stably transfected by the p1.1eGFP plasmid and selected in the presence of 200 nM MTX; p1.1(EBVTR-): transfection by the p1.1(EBVTR-)eGFP plasmid using the same conditions. A. Level of intracellular eGFP in cell populations. Error bars indicate the standard deviation, n = 2. B. Proportion of eGFP-negative cell populations measured by FACS. C. Number of copies of genome-integrated plasmids measured by Q-PCR. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3-4. The apparent level of the eGFP ORF DNA for the untransfected CHO DG44 cells is below 0.1 copies per one haploid genome. D. Codes for the different cell populations and the concentrations of antibiotics employed.

Mentions: Target gene amplification for polyclonal cell populations was performed for the suspension culture of CHO DG44 cells, stably transfected by the p1.1eGFP plasmid in presence of 50 nM MTX, as described above. Concentration of the MTX in the culture medium was increased by two-fold steps, each after two consecutive passages, until the cell viability decreased below 85%. Resulting culture, obtained in presence of 0.8 μM MTX, was split into four flasks, supplemented by 0.8; 1.6; 3.2; 6.4 μM MTX and cultured until the cell viability returned to at least 85% (7–12 days).Generation of polyclonal cell populations involving transfected p1.2 plasmids were performed by seeding transiently transfected cells in 6-well culture plates, using 1 million of viable cells per well in 5 ml of DG44 medium, supplemented with the corresponding antibiotic, or 5 ml of OptiCHO medium with 200 nM MTX for control transfections using p1.1 plasmids. The concentrations of the antibiotics used are shown in Figure 3. Plates were cultivated with shaking until the cell viability returned to at least 85% (20 days), after which the medium was changed every 4 days.


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)

Properties of the cell populations stably transfected by p1.2-based plasmids under various drug selection stringencies. DG44: untransfected CHO DG44 cells; p1.1: cells stably transfected by the p1.1eGFP plasmid and selected in the presence of 200 nM MTX; p1.1(EBVTR-): transfection by the p1.1(EBVTR-)eGFP plasmid using the same conditions. A. Level of intracellular eGFP in cell populations. Error bars indicate the standard deviation, n = 2. B. Proportion of eGFP-negative cell populations measured by FACS. C. Number of copies of genome-integrated plasmids measured by Q-PCR. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3-4. The apparent level of the eGFP ORF DNA for the untransfected CHO DG44 cells is below 0.1 copies per one haploid genome. D. Codes for the different cell populations and the concentrations of antibiotics employed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Properties of the cell populations stably transfected by p1.2-based plasmids under various drug selection stringencies. DG44: untransfected CHO DG44 cells; p1.1: cells stably transfected by the p1.1eGFP plasmid and selected in the presence of 200 nM MTX; p1.1(EBVTR-): transfection by the p1.1(EBVTR-)eGFP plasmid using the same conditions. A. Level of intracellular eGFP in cell populations. Error bars indicate the standard deviation, n = 2. B. Proportion of eGFP-negative cell populations measured by FACS. C. Number of copies of genome-integrated plasmids measured by Q-PCR. Amplicons are located inside the eGFP ORF and one representative value experiment from three independent measurements is shown. Error bars represents standard deviations, n = 3-4. The apparent level of the eGFP ORF DNA for the untransfected CHO DG44 cells is below 0.1 copies per one haploid genome. D. Codes for the different cell populations and the concentrations of antibiotics employed.
Mentions: Target gene amplification for polyclonal cell populations was performed for the suspension culture of CHO DG44 cells, stably transfected by the p1.1eGFP plasmid in presence of 50 nM MTX, as described above. Concentration of the MTX in the culture medium was increased by two-fold steps, each after two consecutive passages, until the cell viability decreased below 85%. Resulting culture, obtained in presence of 0.8 μM MTX, was split into four flasks, supplemented by 0.8; 1.6; 3.2; 6.4 μM MTX and cultured until the cell viability returned to at least 85% (7–12 days).Generation of polyclonal cell populations involving transfected p1.2 plasmids were performed by seeding transiently transfected cells in 6-well culture plates, using 1 million of viable cells per well in 5 ml of DG44 medium, supplemented with the corresponding antibiotic, or 5 ml of OptiCHO medium with 200 nM MTX for control transfections using p1.1 plasmids. The concentrations of the antibiotics used are shown in Figure 3. Plates were cultivated with shaking until the cell viability returned to at least 85% (20 days), after which the medium was changed every 4 days.

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