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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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Relative DNA content of three constructs: pPNT/E-CTCF7-mP that contains the CTCF7 fragment between the promoter and enhancer, pPNT/EmpS-CTCF7 that contains the CTCF7 fragment outside the promoter-enhancer pair, and pPNT/E-l-mp--the control construct with a lambda DNA fragment inserted between the promoter and enhancer in genomic DNA of CHO cells after positive-negative selection. The relative content was estimated by real-time PCR based on the difference between the number of PCR cycles required to detect the target (for detail, see Methods).
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Figure 4: Relative DNA content of three constructs: pPNT/E-CTCF7-mP that contains the CTCF7 fragment between the promoter and enhancer, pPNT/EmpS-CTCF7 that contains the CTCF7 fragment outside the promoter-enhancer pair, and pPNT/E-l-mp--the control construct with a lambda DNA fragment inserted between the promoter and enhancer in genomic DNA of CHO cells after positive-negative selection. The relative content was estimated by real-time PCR based on the difference between the number of PCR cycles required to detect the target (for detail, see Methods).

Mentions: To quantitatively estimate the efficiency of the CTCF binding sequences selection, we pooled together three constructs, namely pPNT/EmP with the CTCF7 fragment inserted between the promoter and enhancer, and two controls-pPNT/EmPS with the CTCF7 fragment inserted outside the promoter-enhancer pair and pPNTE-λ-mP with the lambda phage fragment inserted between the promoter and enhancer. This equimolar mixture was subjected to positive-negative selection procedure described above. Genomic DNAs were then isolated from Neo and ganciclovir resistant cells and used as templates for real-time quantitative PCR. The results are presented in Figure 4.


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)

Relative DNA content of three constructs: pPNT/E-CTCF7-mP that contains the CTCF7 fragment between the promoter and enhancer, pPNT/EmpS-CTCF7 that contains the CTCF7 fragment outside the promoter-enhancer pair, and pPNT/E-l-mp--the control construct with a lambda DNA fragment inserted between the promoter and enhancer in genomic DNA of CHO cells after positive-negative selection. The relative content was estimated by real-time PCR based on the difference between the number of PCR cycles required to detect the target (for detail, see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Relative DNA content of three constructs: pPNT/E-CTCF7-mP that contains the CTCF7 fragment between the promoter and enhancer, pPNT/EmpS-CTCF7 that contains the CTCF7 fragment outside the promoter-enhancer pair, and pPNT/E-l-mp--the control construct with a lambda DNA fragment inserted between the promoter and enhancer in genomic DNA of CHO cells after positive-negative selection. The relative content was estimated by real-time PCR based on the difference between the number of PCR cycles required to detect the target (for detail, see Methods).
Mentions: To quantitatively estimate the efficiency of the CTCF binding sequences selection, we pooled together three constructs, namely pPNT/EmP with the CTCF7 fragment inserted between the promoter and enhancer, and two controls-pPNT/EmPS with the CTCF7 fragment inserted outside the promoter-enhancer pair and pPNTE-λ-mP with the lambda phage fragment inserted between the promoter and enhancer. This equimolar mixture was subjected to positive-negative selection procedure described above. Genomic DNAs were then isolated from Neo and ganciclovir resistant cells and used as templates for real-time quantitative PCR. The results are presented in Figure 4.

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