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Papers In Press, published online ahead of print March 16, 2001
Sidney Kimmel Cancer Center, San Diego, CA 92121
Corresponding Author: braschke{at}skcc.org
Consistent expression from CD45 cDNA constructs has proven difficult to achieve. Through the use of new CD45 cDNA constructs and reporter genes, the role 5', 3' and intron sequences play in CD45 expression was determined. The CD45 polyadenylation signal sequence was fully functional in a b-galactosidase reporter construct. Furthermore, the CD45 3' UTR and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed which contain either the cytomegalovirus (CMV) promoter, the LFA-1 (CD11a) promoter, or various CD45 5' regions. Neither the CMV nor the LFA-1 promoter was capable of generating detectable levels of expression in constructs with CD45 cDNA. However, when CD45 intron sequences between exons 3 and 9 were inserted in the cDNA construct to generate a CD45 minigene, the LFA-1 promoter was able to drive reproducible, significant expression of CD45. CD45 minigenes using the CD45 5' sequences up to 19 kb upstream of the transcriptional start produced very little protein. The LFA-1 CD45 minigene construct produced correct cell type specific isoforms when expressed in T and B lymphocyte lines. Therefore, we conclude that the regulation of CD45 expression and cell type specific splicing requires elements within the intron sequences.
J. Biol. Chem, 10.1074/jbc.M100448200
Submitted on January 17, 2001
Revised on February 26, 2001
Accepted on March 16, 2001
The role of intron sequences in high level expression from CD45 cDNA constructs
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