Mar Characteristic Elements-mediated Episomal Vector Was Found In Mammalian Cells

Backgound The new type of episomal expression vector exists in host cells in form of extrachromosomes, which make transgene expression efficient and continuous. Due to the safety problems related to virus-based epiomes considerable efforts to construct episomal vectors which can overcome the potential danger of insertional mutagenesis that are composed exclusively from functional chromosomal elements have been undertaken within the past decades. Recently more researches focus on S/MAR-mediated episomal vectors, which are combined with matrix attachment region during the mitosis period to make transgene efficient and continuous expression and do not integrate into the genomes in mammalian cells in form of extrachromosomes. Moreover, the episomal vectors exist in host cells at a low copy number and are duplicated to the progeny cells by depending on the cell cycle. MAR components-mediated vectors allow transgene efficient, episomal, and continued. However, the specific mechanism is not very clear so far.Objective To vertify synthetic matrix attachment region (MAR) characteristic sequence mediated vector whether to exist in forms of episome and to drive transgene high expression in transfected mammalian cells.Methods The synthetic MAR characteristic sequences (387bp) were isolated from MAR drived from human β-interferon and then were connected together. The synthesized DNA sequence was inserted into the parental pEGFP-C1to construct vector. pEGFP-C1and reconstructed vector were transfected into Chinese hamster ovary cells (CHO) and mice Neuroblastoma (N-2a) cells. Under the conditions of the pressure choice, the stable monoclonal cells lines were selected respectively. The following was to extract episomal vectors from the stable monoclonal cell lines by modificated Hirt cracking protocol and then was transformed into the host bacteria E.coli DH5a by cacl2method, finally to extract the plasmid once again to carry on plamid rescuing assay. Both rescued plamids and the original plasmid were analyzed by enzyme digestion identification and sequencing analysis simultaneously. The extraction of the genomes and episomal plasmids from the stable monoclonal cell lines were analyzed by PCR. Fluorescence in situ hybridization experiment (FISH) was observed the status of transfected plasmid in mammalian cells. Fluorescence images analyzed the expression level of enhanced green fluorescent protein (EGFP) of both cells through the fluorescent microscope.Results Both rescued plamids and the original plasmid were completely digested by KpnI/BamHI double enzyme and BamHI single enzyme. As a result, it displayed the inserted sequence of387bp bands and linear pEGFP-C1sequences. The episomal plamids extracted from positive group of cells required387bp MAR and598bp bands by PCR amplification. The genome extracted extracted from negative control group cells required598bp band but the episomal plamid did not by PCR amplification. Sequencing analysis confirmed the maps of the rescued plamids were identical to the original DNA sequence, it was shown that the transfected plasmids did not integrate into the host genome, and there was not any change in sequences before and after transfection. FISH showed that transfected plasmid in eukaryotic cells was combined with exchromosomes in forms of episome and existed at a low copy number, about4-10copies/cells. Fluorescence images analysis showed that transgene egfp expressed efficiently and lasted120days or more in CHO cells and N-2a cells.Conclusion S/MAR characteristic sequence-based vector can function as stable episomes in cultured mammalian cells, supporting long-term and effective transgene expression.