Retrovirus-mediated transduction of an engineered intron-containing purine nucleoside phosphorylase gene.
We constructed and tested several retroviral vectors containing abbreviated purine nucleoside phosphorylase (PNP) genes in the reverse orientation, a strategy compatible with transduction of intron-containing genes. We observed two types of deletions in these vectors after one round of replication: (i) Deletions flanked by direct repeats with one copy of the repeat retained in the provirus, presumably resulting from reverse transcriptase slippage during (-) strand DNA synthesis. (ii) Deletions due to fortuitous splice sites in the PNP complementary strand. Two splice donor sites and three splice acceptor sites were identified in a 3.0-kb PNP minigene. We found that the splice donor sites (but not the splice acceptor sites) could be predicted by sequence analysis of the PNP complementary strand. To increase the frequency of intact PNP gene transduction, we introduced sequence modifications: The putative PNP polyadenylation signal and a truncated 117-bp 3' flank were recovered from a rearranged provirus and inserted in place of a 1.2-kb genomic 3' flank. Sequences associated with deletions were eliminated from the PNP 5' untranslated region, and two fortuitous splice donor signals in the complementary strand were inactivated. A retroviral vector LN-PMG11, containing the engineered 2.9-kb PNP minigene in the reverse orientation, was transduced intact in 23% (5/22) of clones after one round of replication and in 87% (20/23) of clones after a second round of replication from two primary virus producer clones. Directed mutagenesis of sequences preventing intact retroviral transduction thus provided a 2.9-kb PNP gene that was transduced intact and expressed at a high level.
Jonsson
J J
JJ
Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455-0392, USA.
Habel
D E
DE
McIvor
R S
RS
D001483:Base Sequence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D007438:Introns; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency / Q000235:genetics*; D012326:RNA Splicing; D012190:Retroviridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D011683:Purine-Nucleoside Phosphorylase