Post-entrapment genome engineering: First exon size does not affect the expression of fusion transcripts generated by gene entrapment

Abstract

Gene trap mutagenesis in mouse embryonic stem cells has been widely used for genome-wide studies of mammalian gene function. However, while large numbers of genes can be disrupted, individual mutations may suffer from limitations due to the structure and/or placement of targeting vector. To extend the utility of gene trap mutagenesis, replaceable 3Ј [or poly(A)] gene trap vectors were developed that permit sequences inserted in individual entrapment clones to be engineered by Cre-mediated recombination. 3Ј traps incorporating different drug resistance genes could be readily exchanged, simply by selecting for the drug-resistance gene of the replacement vector. By substituting different 3Ј traps, we show that otherwise identical fusion genes containing a large first exon (804 nt) are not expressed at appreciably lower levels than genes expressing small first exons (384 and 151 nt). Thus, size appears to have less effect on the expression and processing of first exons than has been reported for internal exons. Finally, a retroviral poly(A) trap (consisting of a RNA polymerase II promoter, a neomycin-resistance gene, and 5Ј-splice site) typically produced mutagenized clones in which vector sequences spliced to the 3Ј-terminal exons of cellular transcription units, suggesting strong selection for fusion transcripts that evade nonsense-mediated decay.

Site‐directed Mutagenesis

“…This provides further evidence that poly(A) traps select for fusion transcripts that evade nonsense-mediated decay (28,38). However, the preference was less pronounced, as 29% of the inserts in wellcharacterized genes were in upstream introns.…”

“…However, like other early-generation poly(A) traps, the vector preferentially targets 3 0 introns, reflecting selection for fusion transcripts that escape nonsense-mediated decay (28,38). This interferes with the mutagenesis of a subset of genes that lack significant protein coding sequence in the last exon and may limit genome coverage (40).…”

“…Expression of the occupied cellular genes is disrupted by a 3 0 exon consisting of sequences from the 3 0 end of a puromycin-resistance gene, an internal ribosome entry site (IRES) and a reporter protein [either a nuclear b-galactosidase (lacZ; GTR1.0-GTR1.3) or enhanced green fluorescent protein (EGFP; GTR1.4-GTR1.7)]. Wild-type and mutant [lox5171, (35)] loxP sites allow provirus inserts to be engineered by recombinasemediated cassette exchange (RMCE) (27)(28)(29). GTR1.0 and GTR1.3 contain an additional loxP5171 site located in a synthetic intron inserted into the Neo gene ( Supplementary Figure 1c).…”

Novel Radiation Response Genes Identified in Gene-Trapped MCF10A Mammary Epithelial Cells

“…In contrast, we found the majority of genes to be down-regulated after irradiation. We believe that the poly-A trap retroviral vector was preferentially targeting genes with smaller first exons due to the addition of the neomycin reporter construct (71)(72)(73)(74). This may explain why only five genes were identified out of the initial 31 clones that were changed at least twofold after radiation treatment.…”

Genomewide production of multipurpose alleles for the functional analysis of the mouse genome

“…Moreover, effective gene trapping is restricted to the Ϸ70% of the genes expressed in ES cells (27,28). Although gene trapping strategies have been described for genes that are not expressed in ES cells, their performance is quite inconsistent, making them unsuitable for high-throughput approaches (29)(30)(31). We believe that for a comprehensive mutagenesis of the mouse genome, a balance between gene trapping and gene targeting, performed with generic gene trap cassettes inserted into the targeting vectors, is likely to be the most efficient and cost-effective.…”