BackgroundThe hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing.Results In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression.ConclusionVectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.
Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure vari-
The quaternary structure of the adenovirus early region 1B 54K protein has been examined under denaturing and nondenaturing conditions. In the presence of SDS the protein has a strong tendency to form disulfide-linked high-molecular-weight polymers. Under nondenaturing, but reducing, conditions the in vitro-translated 54K polypeptide forms oligomers (probably tetramers) of molecular weight approximately 2 x 10(5). After subcellular fractionation of Ad12 early region 1-transformed cells, the 54K E1B protein present in the cytoplasm had a molecular weight similar to that determined for the in vitro-translated material. However, two populations of the viral protein could be distinguished in the nucleus-one of a size similar to that seen in the cytoplasm and the other of appreciably higher molecular weight (approximately 2 x 10(6)). No difference in migration pattern was observed after treatment of the nuclear extract with DNase I or RNase. A proportion of the Ad12 E1B 54K protein in both the high- and the low-molecular-weight populations in the nucleus was found to form a complex with p53, and it is therefore concluded that the very high molecular weight derives from interaction with an, as yet, unidentified component.
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