Author
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REILLY, J - MICHIGAN STATE UNIV |
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Silva, Robert |
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Submitted to: DNA and Cell Biology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 5/31/1996 Publication Date: N/A Citation: N/A Interpretive Summary: The use of molecular biological techniques offers some promise of generating new and more efficacious vaccines. However, the insertion of foreign genes (the basic unit of DNA that encodes the information for a protein) into a virus in order to generate recombinant vaccines often results in the mutation of one or more genes that adversely affects vaccine virus replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes consist of short pieces of DNA that were derived from an intron (a region within a gene that does not code for proteins and is removed from the gene by the normal cellular enzymatic machinery). When inserted into some test genes, we were able to show that the introns were correctly removed and the test genes functioned as well as test genes without the intron. To demonstrate the usefulness of our portable introns, we inserted one test gene within an intron and then inserted the intron/test gene construct within a second test gene. Both genes were functional, demonstrating that neither gene was mutated nor adversely affected by the newly added intron sequences. This is the first report of a technique that allows one gene to be inserted into another gene and still allow both genes to be functional. A modified portable intron should be valuable in situations where it is necessary to deliver foreign genes into sites where it is important to not disrupt existing genes. Some possible applications include gene therapy and the development of new recombinant DNA vaccines. Technical Abstract: The insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affects replication. In an attempt to overcome this problem, we constructed two portable intron cassettes. The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli. The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase or CAT activity. The first intron cassette (type A) contained flanking adenovirus exon sequences. Consequently, the flanking adenovirus exon sequences remained in the spliced transcript. With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites, PmlI and PvuII, respectively. The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene. After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript. To demonstrate its usefulness, an insertion cassette was made by cloning the E. coli LacZ gene into a multiple cloning site within the type B intron. The insertion cassette was then cloned into a PvuII site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-galactosidase were detected, demonstrating that both genes were properly transcribed and translated. |
