Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome / 中国药科大学学报

@#Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.

Analysis on Immune Effect of 10 Microgramme Recombinant Chinese Hamster Ovary (CHO) Cell HB Vaccine in Adults / 医学研究杂志

Objective To explore immune effects of HB vaccine and relative strategies of controlling hepatitis B in adults of different ages, genders, and different anti-HBs levels before immunization. Methods In Shaoxing city, 1055 healthy adults aged 18 to 50 with HBsAg negative were randomly selected and inoculated with 10?g/ml indigenous CHO HB vaccine according to the programme of 0,1,6 months. Results 835 adults completed the whole programme, and the total immunization coverage rate was 85.12%. Before and after immunization,the positive rates of anti-HBs were 52.10% and 98.44% respectively, and anti-HBsGMT rose from 14.32mU/ml to 413.98mU/ml. For the two groups, anti-HBsGMT=0 and 0

Protective effects of active fraction and constituents from Flos Daturae on Chinese hamster ovary cells injuried by dimethyl sulfoxide / 中草药

Objective To observe the protective effects of various extracts from Flos Daturae (FD), including active fraction (AF-FD), withanolides constituents (WC-FD), and flavonoids constituents (FC-FD) on Chinese hamster ovary (CHO) cells against cytotoxicity induced by DMSO. Methods The survival rate of CHO cells was examined by MTT assay and LDH leakage assays. Results Using MTT assay, coincubation of CHO cells with 3% DMSO for 24 h resulted in a significant reduction of survival rate of CHO cells. AF-FD was tested in a range of 10—80 ?g/mL to improve the survival rate of CHO cells in a dose-dependent manner. FC-FD (2.5—20 ?g/mL), but not WC-FD (30—120 ?g/mL), could significantly relieve the injury induced by 3% DMSO in CHO cells. In the measurement of LDH leakage, coincubation of CHO cells with 4.5% DMSO for 24 h obviously increased LDH release. However, all the compounds tested, including AF-FD (10—80 ?g/mL), WC-FD (30—120 ?g/mL), and FC-FD (2.5—20 ?g/mL) had no effect on LDH leakage induced by 4.5% DMSO. Conclusion The findings suggest that the FC-FD may protect CHO cells from DMSO cytotoxicity assessed by MTT assay, which may be associated with improving mitochondrial function, but not protecting the membrane injury of CHO cells.

Cloning and expression of rat liver type glucose transporter and translocation by insulin in Chinese hamster ovary cells

The 5'- and 3'-side half of liver type glucose transporter (GLUT2) cDNA was amplified from total RNA or mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified 5'-side fragment of GLUT2 cDNA was inserted into pGEM4Z and named pGLGT1, and the 3'-side fragment of GLUT2 cDNA was inserted into the HindIII site of pGLGT1 to construct pGLGT2 which contains an entire open reading frame of GLUT2 cDNA. The GLUT2 cDNA in pGLGT2 was transferred to an eukaryotic expression vector (pMAM) to construct pMLGT, which was expressed in the insulin-sensitive Chinese hamster ovary (CHO) cells. Western blot analysis showed that the GLUT2 gene in pMLGT was expressed in the transfected CHO cells successfully. The GLUT2 content in the plasma membrane fraction of insulin-treated CHO cells expressing GLUT2 increased 3.8-fold compared to that of the control group. This result suggests that GLUT2, which is not subjected to translocation by insulin in the cells of its major distribution, can be translocated if it is expressed in the suitable cells sensitive to insulin action.