Sequence analysis of scaffold/matrix attachment regions (S/MARs) from human embryonic kidney and Chinese hamster ovary cells

Nur Shazwani Mohd Pilus, Azrin Ahmad, Nurul Yuziana Mohd Yusof

Research output: Contribution to journalArticle

Abstract

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 (Sequence raw data from this article have been deposited at the EMBL Data Libraries under Study ID PRJEB26090 (ERP108063)). 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.

Original languageEnglish
Pages (from-to)387-400
Number of pages14
JournalOnLine Journal of Biological Sciences
Volume18
Issue number4
DOIs
Publication statusPublished - 1 Jan 2018

Fingerprint

Matrix Attachment Regions
Chinese hamsters
Cricetulus
Scaffolds
Sequence Analysis
Ovary
sequence analysis
Cells
kidneys
Kidney
cells
Genes
methodology
cell lines
nuclear matrix
genome
lithium
consensus sequence
Nuclear Matrix-Associated Proteins
recombinant proteins

Keywords

  • 2M NaCl
  • Biopharmaceutical
  • Expression vector
  • Gene transcription
  • Intergenic
  • LIS

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Sequence analysis of scaffold/matrix attachment regions (S/MARs) from human embryonic kidney and Chinese hamster ovary cells. / Pilus, Nur Shazwani Mohd; Ahmad, Azrin; Mohd Yusof, Nurul Yuziana.

In: OnLine Journal of Biological Sciences, Vol. 18, No. 4, 01.01.2018, p. 387-400.

Research output: Contribution to journalArticle

@article{f231784114084a45b8254685b8deaf88,
title = "Sequence analysis of scaffold/matrix attachment regions (S/MARs) from human embryonic kidney and Chinese hamster ovary cells",
abstract = "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 (Sequence raw data from this article have been deposited at the EMBL Data Libraries under Study ID PRJEB26090 (ERP108063)). 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.",
keywords = "2M NaCl, Biopharmaceutical, Expression vector, Gene transcription, Intergenic, LIS",
author = "Pilus, {Nur Shazwani Mohd} and Azrin Ahmad and {Mohd Yusof}, {Nurul Yuziana}",
year = "2018",
month = "1",
day = "1",
doi = "10.3844/ojbsci.2018.387.400",
language = "English",
volume = "18",
pages = "387--400",
journal = "OnLine Journal of Biological Sciences",
issn = "1608-4217",
publisher = "Science Publications",
number = "4",

}

TY - JOUR

T1 - Sequence analysis of scaffold/matrix attachment regions (S/MARs) from human embryonic kidney and Chinese hamster ovary cells

AU - Pilus, Nur Shazwani Mohd

AU - Ahmad, Azrin

AU - Mohd Yusof, Nurul Yuziana

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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 (Sequence raw data from this article have been deposited at the EMBL Data Libraries under Study ID PRJEB26090 (ERP108063)). 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.

AB - 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 (Sequence raw data from this article have been deposited at the EMBL Data Libraries under Study ID PRJEB26090 (ERP108063)). 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.

KW - 2M NaCl

KW - Biopharmaceutical

KW - Expression vector

KW - Gene transcription

KW - Intergenic

KW - LIS

UR - http://www.scopus.com/inward/record.url?scp=85061985534&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85061985534&partnerID=8YFLogxK

U2 - 10.3844/ojbsci.2018.387.400

DO - 10.3844/ojbsci.2018.387.400

M3 - Article

VL - 18

SP - 387

EP - 400

JO - OnLine Journal of Biological Sciences

JF - OnLine Journal of Biological Sciences

SN - 1608-4217

IS - 4

ER -