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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

PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to the sns insulator in direct (+) or reverse (-) orientation relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).
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Figure 2: PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to the sns insulator in direct (+) or reverse (-) orientation relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).

Mentions: After the positive-negative selection, genomic DNA was isolated from the pPNT/E-sns-mP transfected cells and used to determine the presence and orientation of the sns insulator inserts by PCR amplification (Figure 2). The primer pairs for amplification of the sns insulator in both orientations and their sequences are presented in Tables 1 and 2. As seen from Figure 2, the PCR produced DNA fragments of the expected lengths, which means that the HSV-tk expression in the transfected cells was suppressed or significantly reduced, and that the sns element in CHO cells was active as enhancer blocker in both orientations.


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)

PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to the sns insulator in direct (+) or reverse (-) orientation relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to the sns insulator in direct (+) or reverse (-) orientation relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).
Mentions: After the positive-negative selection, genomic DNA was isolated from the pPNT/E-sns-mP transfected cells and used to determine the presence and orientation of the sns insulator inserts by PCR amplification (Figure 2). The primer pairs for amplification of the sns insulator in both orientations and their sequences are presented in Tables 1 and 2. As seen from Figure 2, the PCR produced DNA fragments of the expected lengths, which means that the HSV-tk expression in the transfected cells was suppressed or significantly reduced, and that the sns element in CHO cells was active as enhancer blocker in both orientations.

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