CONSTRUCTION OF PORTABLE INTRON CASSETTES FOR THE DELIVERY AND EXPRESSION OF FOREIGN GENES

The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines, However, the insertio n of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication. I n an attempt to overcome this problem, we constructed two portable int ron cassettes, The cassettes were derived from the adenovirus late lea der 1 intron and were cloned into either the chloramphenicol acetyltra nsferase (CAT) gene or the LacZ gene of Escherichia coli, The intron c assettes were transfected into chicken embryo fibroblasts (CEFs) and t he cell lysates were later assayed for either beta-galactosidase (beta -Gal) or CAT activity, The first intron cassette (type A) contained fl anking adenovirus exon sequences, Consequently, the flanking adenoviru s exon sequences remained in the spliced transcript, With the type A i ntron inserted in the correct orientation for splicing, CAT activity w as not diminished, However, in the reverse orientation, no CAT activit y could be detected, The second intron cassette (type B) had the splic e donor and splice acceptor sites converted to the blunt-end restricti on endonuclease sites Pml I and Pvu II, respectively, The blunt-end re striction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blu nt-end restriction endonuclease site in the recipient gene, After spli cing, no adenovirus exon sequences remained in the recipient gene's RN A transcript, To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site wi thin the type B intron, The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene, Following transfection in C EFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.