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DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity.

Didych DA, Kotova ES, Akopov SB, Nikolaev LG, Sverdlov ED - BMC Res Notes (2012)

Bottom Line: Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya, Moscow 117997, Russia.

ABSTRACT

Background: Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.

Results: Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.

Conclusions: We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

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Related in: MedlinePlus

Plasmid constructions used in this work. mPGK-1-mouse phosphoglycerate kinase I promoter; NeoR-E. coli neomycin phosphotransferase gene; HSV-tk-herpes simplex virus thymidine kinase gene. Broken arrows indicate direction of transcription. For detailed description, see the text.
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Figure 1: Plasmid constructions used in this work. mPGK-1-mouse phosphoglycerate kinase I promoter; NeoR-E. coli neomycin phosphotransferase gene; HSV-tk-herpes simplex virus thymidine kinase gene. Broken arrows indicate direction of transcription. For detailed description, see the text.

Mentions: The constructs obtained are shown in Figure 1. Prior to electroporation, the constructs were linearized with Eco47 III (Fermentas).


DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity.

Didych DA, Kotova ES, Akopov SB, Nikolaev LG, Sverdlov ED - BMC Res Notes (2012)

Plasmid constructions used in this work. mPGK-1-mouse phosphoglycerate kinase I promoter; NeoR-E. coli neomycin phosphotransferase gene; HSV-tk-herpes simplex virus thymidine kinase gene. Broken arrows indicate direction of transcription. For detailed description, see the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369819&req=5

Figure 1: Plasmid constructions used in this work. mPGK-1-mouse phosphoglycerate kinase I promoter; NeoR-E. coli neomycin phosphotransferase gene; HSV-tk-herpes simplex virus thymidine kinase gene. Broken arrows indicate direction of transcription. For detailed description, see the text.
Mentions: The constructs obtained are shown in Figure 1. Prior to electroporation, the constructs were linearized with Eco47 III (Fermentas).

Bottom Line: Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya, Moscow 117997, Russia.

ABSTRACT

Background: Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.

Results: Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.

Conclusions: We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

Show MeSH
Related in: MedlinePlus