SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

Abstract

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. 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. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. 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.

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

“…Several stimulating introns have been identified in other model systems which originate from viral DNA or different eukaryotic genomes including plants, fungi, and mammals [54][55][56][57][58][59]. Although their mode of action is not fully elucidated, typically, these introns are applied in genetic engineering strategies as synthetic insertions in transformation constructs or promoters [60].…”

In vivo measurements reveal a single 5′-intron is sufficient to increase protein expression level in Caenorhabditis elegans

“…We compare the size distribution of introns using modern drafts of the C. elegans and human genomes. Understanding details of IME is important for basic biology 36 , biotechnology 37,38 , and human diseases. There are now several diseases in which mutations in introns affect gene regulation 39,40 .…”

In vivomeasurements reveal a single 5’ intron is sufficient to increase protein expression level inC. elegans

“…Here, we are focused on IME of gene expression in order to: 1) quantify the magnitude that introns increase protein expression levels in C. elegans, and 2) to develop C. elegans as an in vivo, genetically tractable, screenable model to help reveal the many different ways that introns act to influence the functions of biological systems. This understanding may be particularly important not just for basic biology (YOFE et al 2014) and biotechnology (LACY-HULBERT et al 2001;XU et al 2018), but for human diseases, including those where intronic mutations affect gene regulation (RILEY AND KRIEGER 2005;VAZ-DRAGO et al 2017). Thus, employing C. elegans to understand the basic biology of introns using in vivo measurements of reporter proteins may offer novel insights into the physiology and molecular biology of introns, including IME of gene expression levels, and the ways that allelic variants of introns affect gene expression -both cis and trans.…”