The role of intron sequences in high level expression from CD45 cDNA constructs.
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 beta-galactosidase reporter construct. Furthermore, the CD45 3'-untranslated region and downstream sequences were shown to contain no negative regulatory elements. Several new CD45 cDNA constructs were designed that contain either the cytomegalovirus promoter, the leukocyte function-associated antigen (LFA-1; CD11a) promoter, or various CD45 5' regions. Neither the cytomegalovirus 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 kilobases 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.
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Sidney Kimmel Cancer Center, San Diego, California 92121, USA.
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D020413:3' Untranslated Regions; D018076:DNA, Complementary; D006801:Humans; D007438:Introns; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000235:genetics*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D016169:Lymphocyte Function-Associated Antigen-1; D017493:Leukocyte Common Antigens