BioProject

Binding of intergenic scaffold/matrix attachment regions (S/MARs) to nuclear matrix proteins is believed to poise adjacent genes for transcription by forming chromatin loops. More...

Binding of intergenic scaffold/matrix attachment regions (S/MARs) to nuclear matrix proteins is believed to poise adjacent genes for transcription by forming chromatin loops. Vector constructs containing scaffold/matrix attachment regions (S/MAR) flanking the gene of interest, therefore, are able to enhance recombinant protein expression in mammalian cells. We compared two methods that are based on buffers containing 2M NaCl and lithium-3,5-diidosalicylate (LIS) to isolate S/MARs from HEK293 and CHO DG44 cell lines. Isolated S/MARs were sequenced using the Illumina HiSeq platform and mapped against CHO DG44 genome contigs and the human genome GRCh37.p13 respectively. The 2M NaCl method produced 16 million S/MAR consensus sequences which included nine million and seven million from HEK293 and CHO DG44 respectively. LIS method, on the other hand, generated thirteen million S/MAR consensus containing 8.4 million and 4.7 million from HEK293 and CHO DG44, respectively. In order to compare all sets of S/MAR consensus, BLASTN analyses were performed based on exact matches. The number of perfect matches between S/MAR sequences produced by both methods was quite low: 0.46% and 0.07% for HEK293 and CHO DG44 cells respectively, indicating that the two methods isolate different sets of S/MARs. Comparison between the two cell lines found six S/MARs in common, with average coverage of 82%, obtained by the 2M NaCl method, but none of these are intergenic. The LIS method gave 38 S/MARs with average coverage of 85%, common to both cell types; of these, 13 were intergenic. We hypothesize that S/MARs from HEK293 and CHO DG44 isolated using the LIS method have the potential to be universal vector expression elements that can overcome the problem of low production yield. Less...