Limits...
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.

Show MeSH

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 10 CTCF-binding DNA fragments. A-"direct", and B-"reverse" orientation of the fragments relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3369819&req=5

Figure 3: PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to 10 CTCF-binding DNA fragments. A-"direct", and B-"reverse" orientation of the fragments relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).

Mentions: The genomic DNA was used as a template for nested PCR. At the first stage, the fragments located between the CMV promoter and enhancer were amplified with primers P1L and P1R (Figure 1). The PCR product contained a mixture of selected CTCF-binding fragments flanked by short fragments of the pPNT/EmP DNA. This mixture was used as a template for the second PCR round with internal primers specific for each CTCF-binding fragment and the control sns insulator (Table 2). Each individual internal primer was used in combination with either P1L or P1R in order to determine both the presence and orientation of the CTCF-binding fragments in the selected DNA. The results of nested PCR are presented in Figure 3. As seen from Figure 3A,B (upper panels), all 10 CTCF-binding fragments and the control sns insulator were present in the genomic DNA after G418 selection suggesting that the corresponding constructs were inserted into the cellular genome. The same fragments were revealed also after selection with 10 uM (Figure 3A,B, lower panels) or 4 uM ganciclovir (not shown). Therefore, it can be concluded that all 10 fragments which bind CTCF in vitro make the cells resistant to ganciclovir when placed between enhancer and promoter.


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 10 CTCF-binding DNA fragments. A-"direct", and B-"reverse" orientation of the fragments 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 3: PCR products obtained using a genomic DNA template from transfected CHO cells after positive (G418) and positive-negative (Ganciclovir) selection and primers specific to 10 CTCF-binding DNA fragments. A-"direct", and B-"reverse" orientation of the fragments relative to the CMV minimal promoter. M-DNA length marker (SibEnzyme).
Mentions: The genomic DNA was used as a template for nested PCR. At the first stage, the fragments located between the CMV promoter and enhancer were amplified with primers P1L and P1R (Figure 1). The PCR product contained a mixture of selected CTCF-binding fragments flanked by short fragments of the pPNT/EmP DNA. This mixture was used as a template for the second PCR round with internal primers specific for each CTCF-binding fragment and the control sns insulator (Table 2). Each individual internal primer was used in combination with either P1L or P1R in order to determine both the presence and orientation of the CTCF-binding fragments in the selected DNA. The results of nested PCR are presented in Figure 3. As seen from Figure 3A,B (upper panels), all 10 CTCF-binding fragments and the control sns insulator were present in the genomic DNA after G418 selection suggesting that the corresponding constructs were inserted into the cellular genome. The same fragments were revealed also after selection with 10 uM (Figure 3A,B, lower panels) or 4 uM ganciclovir (not shown). Therefore, it can be concluded that all 10 fragments which bind CTCF in vitro make the cells resistant to ganciclovir when placed between enhancer and promoter.

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