![]() 5 Additionally, DSBs are caused by cancer treatment procedures such as radiotherapy and chemotherapy. 3, 4 DSBs result from physiological processes, including V(D)J recombination and meiosis, which generate DSBs as intermediates. 2 The incorporation of errors during DSB repair may lead to chromosomal rearrangements, such as translocation and deletion, which lead to proto-oncogenic transformations or cell death. DNA double-strand breaks (DSBs) are considered to be the most lethal type of DNA damage in cells. 1 Typically, genomic integrity is protected by a series of processes, including DNA damage repair, cell cycle checkpoints, and apoptosis. Several DNA repair pathways work to protect the genome from genotoxic stresses. Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins.Maintenance of genome integrity is important for the survival of all organisms. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT-PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections.įurthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. ![]() In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in CHO cells.Ĭhinese hamster ovary (CHO) gene expression gene regulation intron. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.Įffect of different introns on the production of eGFP in stable transfected CHO‐S cell. ( A) Relative eGFP mRNA levels in stable transfected cells. At 48 hrs after transfection, stable transfected cells were selected by 800 μg/ml geneticin selection for 2 weeks until positive colonies appeared. Then, stable cell populations exhibiting stable transgene integration were cultured in CD CHO medium supplemented with 8 mM l‐glutamine in 125‐ml Corning shake flasks with 30 ml medium with the presence of 500 μg/ml G418 for 30 days. Total RNA was isolated from 5 × 10 6 cells using the RNApure Tissue Kit, analysis of the mRNA levels was determined using real‐time quantitative PCR. ( B) eGFP MFI was determined by cytometry for stable transfected cell pool with the different introns. Cells were collected and the eGFP fluorescence profile was measured for by FACSCalibur at 30 days after transfection. ( C) eGFP MFI expression was normalized to IVS, and in the statistical analysis of eGFP expression, fold change values were normalized to those of the IVS, whose value was set to 1. Standard error of the mean (S.E.M.) is indicated (Student's t‐test, * P < 0.05). ( D) Percentage of high producers (% M3), fluorescence values greater than 10 4. Three independent experiments were performed in this study. Standard error of the mean (S.E.M.) is indicated. ![]() ![]() CV values are expected to reflect variations in transgene expression. The results are the mean values obtained for 3 independent experiments, standard deviation is indicated. ![]() ( F) Correlation between the relative eGFP mRNA levels and relative eGFP protein expression in stable transfected CHO cell. Little correlation was noted between the eGFP mRNA levels and the eGFP protein.Transcripts originating from the SV40 late promoter of pSV2-neo or pSV2-cat contain pBR322 sequences and are polyadenylated at the SV40 late poly(A) site, resulting in an RNA of 3500 nt. ![]()
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