Oviding him with an International Fellowship (ICAR-IF), as von Hippel-Lindau (VHL) Compound partial support of
Oviding him with an International Fellowship (ICAR-IF), as partial help of his PhD research. This function was supported by the United StatesIsrael Binational Agricultural Investigation and Development Fund (BARD) [grant no. US-4571-12C to SM, MLT, and SP-H], and the Chief Scientist of the Israeli Ministry of Agriculture Fund [grant no. 203-0898-10 to SM and SP-H].
Improved elongation factor-1 alpha-based vectors for steady high-level expression of heterologous proteins in Chinese hamster ovary cellsOrlova et al.Orlova et al. BMC Biotechnology 2014, 14:56 biomedcentral.com/1472-6750/14/Orlova et al. BMC Biotechnology 2014, 14:56 biomedcentral.com/1472-6750/14/METHODOLOGY ARTICLEOpen AccessImproved elongation factor-1 alpha-based vectors for steady high-level expression of heterologous proteins in Chinese hamster ovary cellsNadezhda A Orlova1,two, Sergey V Kovnir1,two, Julia A Hodak1,two, Ivan I Vorobiev1,2*, Alexandre G Gabibov2,three and Konstantin G SkryabinAbstractBackground: Establishing highly productive clonal cell lines with constant productivity over 2 months of continuous culture remains a tedious activity requiring the screening of tens of a huge number of clonal colonies. Moreover, long-term cultivation of several candidate lines derived in the absence of drug selection pressure is vital. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene as well as the dihydrofolate reductase (DHFR) choice marker (with separate promoters) may be made use of to get highly productive populations of stably transfected cells within the choice medium, but they haven’t been tested for their ability to help target gene amplification beneath steadily rising methotrexate pressure. Results: We’ve modified EEF1A-based vectors by linking the DHFR selection marker for the target gene in the bicistronic RNA, shortening the all round plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence on the EBVTR element elevated the price of stable transfection by the plasmid by 24 times that with the EBVTR-minus manage and enhanced the rate of methotrexate-driven gene amplification. The mean expression PKD2 custom synthesis degree of the enhanced green fluorescent protein (eGFP) utilised herein as a model protein, increased as much as eight-fold working with a single round of amplification inside the case of adherent colonies formation and as much as 4.5-fold within the case of suspension polyclonal cultures. Numerous eGFP-expressing cell populations created employing vectors with antibiotic resistance markers in place of the DHFR marker were compared with each other. Steady 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 in the total cytoplasmic protein, with much less than five in the cell population getting eGFP-negative. Conclusions: The p1.1 vector was very helpful for steady transfection of CHO cells and capable of rapid MTX-driven target gene amplification, although p1.2-Hygro accomplished comparable eGFP expression levels as p1.1. The set of vectors we’ve created need to speed-up the procedure of producing highly productive clonal cell lines while substantially decreasing the connected experimental work. Keyword phrases: CHO cells, High level expression, Steady cell line generation, Molecular cloning* Correspondence: [email protected] 1 Laboratory of Mammalian Cell Bioengineering, Centre “Bioengineering”, Russian Academy of Sciences, 60-letija Oktyabrya 7, Mosc.
