Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

Osipovich AB, White-Grindley EK, Hicks GG, Roshon MJ, Shaffer C, Moore JH, Ruley HE
Nucleic Acids Res. 2004 32 (9): 2912-24

PMID: 15155860 · PMCID: PMC419606 · DOI:10.1093/nar/gkh604

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

MeSH Terms (17)

Animals Cell Line Exons Genetic Vectors Heterogeneous-Nuclear Ribonucleoprotein Group C Introns Kanamycin Kinase Molecular Sequence Data Mutagenesis, Insertional Mutagenesis, Site-Directed Protein Biosynthesis Proviruses Recombinant Fusion Proteins RNA, Messenger RNA Splice Sites RNA Splicing Terminal Repeat Sequences

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