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. 2008 Nov;36(20):e133.
doi: 10.1093/nar/gkn603. Epub 2008 Sep 23.

Enhanced gene trapping in mouse embryonic stem cells

Frank Schnütgen  1 , Jens Hansen, Silke De-Zolt, Carsten Horn, Marcus Lutz, Thomas Floss, Wolfgang Wurst, Patricia Ruiz Noppinger, Harald von Melchner
Affiliations

Enhanced gene trapping in mouse embryonic stem cells

Frank Schnütgen et al. Nucleic Acids Res. 2008 Nov.

Abstract

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.

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Figures

Figure 1.
Figure 1.
Graphic illustration of enhanced gene trapping. (A) Mechanism of enhanced gene trap activation. Insertion of the osteopontin enhancer (OPE) into an intron of a gene via a gene trap vector activates the endogenous promoter by binding the transcription factor Oct4. The induced promoter sets off the expression of the gene trap cassette, resulting in a productive gene trap event. (B) Enhanced conditional gene trap vector –‘eFlipRosaβgeo’. E, exon; OPE, osteopontin enhancer elements; SA, splice acceptor; pA, polyadenylation sequence; LTR, long terminal repeat; frt/F3, heterotypic FLPe recombinase target sequences; loxP/lox5171, heterotypic Cre recombinase target sequences; βgeo, β-galactosidase/neomycinposphotransferase fusion gene.
Figure 2.
Figure 2.
Induction of gene expression by the osteopontin enhancer (OPE). (A) Top: Schematic representation of the trapped allele before (with enhancer) and after (without enhancer) FLPe- and Cre-mediated recombination. Positions of primers used for the allele specific PCRs are indicated by arrows. Bottom: Allele specific PCR products resolved on 1% agarose gel stained with ethidium bromide. (B) Top: Quantitative RT-PCR analysis of fusion transcripts using gene- and βgeo-specific primers. Results represent the mean relative expression levels ± SD from three separate experiments. Bottom: Oct4 expression in the trapped and recombined ESC lines. Total protein lysates from ESC lines were subjected to SDS–PAGE and analyzed by western blotting using anti-mouse Oct4 and tubulin antibodies. Note that Oct4 expression does not significantly change in the recombined subclones, indicating that the cells are still undifferentiated.
Figure 3.
Figure 3.
5′-RACE efficiency in trapped ESC lines. The fraction of trapped ESC cell lines for which both SPLK-tag and a RACE tag were obtained is shown for 645 eFlipRosaβgeo and 983 FlipRosaβgeo insertions.
Figure 4.
Figure 4.
Oct4 binding by the OPE in trapped ESCs. Chromatin immunoprecipitation was performed as described in the Methods section using anti-Oct4 and anti-V5 antibodies. PCRs of the precipitated and purified DNAs were performed with osteopontin gene- and gene-trap-specific primers. P048G09, parental clone with enhancer; P048G09 G1, recombined subclone without enhancer; MW, molecular weight standard (1 kb plus ladder, Invitrogen).
Figure 5.
Figure 5.
Transcriptional analysis of X-linked mutations in eFlipRosaβgeo trapped ESC lines (also see Supplementary Table 1). (A) Top: RT-PCR products obtained from fusion transcripts expressed in the trapped ESC lines resolved on a 1% agarose gel stained with ethidium bromide. Amplification reactions were performed using trapped gene- and βgeo- specific primers. The double bands in lane 1 correspond to alternative N-terminal splice variants of the ATRX gene. Bottom: RT-PCR products obtained from wild-type transcripts expressed in trapped and parental E14-ESCs resolved on a 1% agarose gel stained with ethidium bromide. Gene-specific primers were chosen in exons flanking the intron containing the insertion. Note the complete absence of detectable wild-type transcripts in the trapped ESC clones. (B) Quantitative RT-PCR of the wild-type transcripts shown in A. Results are derived from triplicate reactions and were normalized to corresponding gene expression levels in E14 parental cells (=100%).
Figure 6.
Figure 6.
Gene trapping efficiencies with enhanced vectors. (A) Overall trapping efficiency. The number of novel genes trapped was plotted against the number of accumulating gene trap sequence tags (GTSTs). (B) Trapping rate of hard to trap genes (httgs). ‘Hard-to-trap genes’ are defined as genes that have no entry or only one entry in the IGTC, Omnibank I or Omnibank II gene trap libraries. The number of hard-to-trap ENSEMBL genes trapped was plotted against the cummulative number of unique genes trapped. Note that only ENSEMBL protein-coding genes were considered for this analysis. (C) Trapping rate of antisense transcripts and intergenic regions. Results are based on SPLK tags obtained from a minimum of 1500 ECS lines trapped with each vector.
Figure 7.
Figure 7.
Box-Plot of absolute gene expression levels of genes trapped with the FlipRosaβgeo vectors. Gene expression values for trapped ENSEMBL genes were derived from the GEO series accession GSE8128 (9). Box boundaries represent the first and third quartiles (Q.25, Q.75). The median is indicated by the horizontal line dividing the interquartile range. Upper and lower ticks indicate the 10th and 90th percentiles. The mean value is indicated by the dashed line (also see Supplementary Table 1).
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