title: DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity.
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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya, Moscow 117997, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Didych
authors.forename: Elena S
authors.initials: ES
authors.lastname: Kotova
authors.forename: Segey B
authors.initials: SB
authors.lastname: Akopov
authors.forename: Lev G
authors.initials: LG
authors.lastname: Nikolaev
authors.forename: Eugene D
authors.initials: ED
authors.lastname: Sverdlov
mesh_terms: D000818:Animals; D000076246:CCCTC-Binding Factor; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015774:Ganciclovir / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D009355:Neomycin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D004247:DNA; D013937:Thymidine Kinase; D009355:Neomycin; D015774:Ganciclovir