Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.
An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells.
Watanabe
Satoshi
S
Aminal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-8602, Japan. kettle@affrc.go.jp
Watanabe
Sachiko
S
Sakamoto
Naoaki
N
Sato
Masahiro
M
Akasaka
Koji
K
D000818:Animals; D001192:Arylsulfatases / Q000235:genetics*; D000076246:CCCTC-Binding Factor; D002478:Cells, Cultured; D016373:DNA, Antisense / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D038101:Insulator Elements / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D012097:Repressor Proteins / Q000235:genetics; D012617:Sea Urchins / Q000235:genetics*; D019076:Transgenes
D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D016373:DNA, Antisense; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D008156:Luciferases; D001192:Arylsulfatases