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 insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect 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 (beta-Gal) 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. However, in the reverse orientation, no CAT activity could be detected. The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, 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 Pvu II site in the middle of the CAT gene. Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated.
Reilly
J D
JD
Department of Animal Science, Michigan State University, East Lansing 48824, USA.
Silva
R F
RF
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D005347:Fibroblasts; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D012326:RNA Splicing / Q000235:genetics*; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase