High-throughput trapping of secretory pathway genes in mouse embryonic stem cells - PubMed (original) (raw)

High-throughput trapping of secretory pathway genes in mouse embryonic stem cells

Silke De-Zolt et al. Nucleic Acids Res. 2006.

Abstract

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.

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Figures

<b>Figure 1</b>

**Figure 1

Schematic representation of the secretory gene trap vectors. LTR, long terminal repeat; Ceo, CD2/neomycinphosphotransferase fusion gene; SA, splice acceptor; TM, transmembrane domain; frt (yellow triangles) and F3 (green triangles) heterotypic target sequences for FLPe recombinase; loxP (red triangle) and lox511 (purple triangles), heterotypic target sequences from Cre recombinase.

<b>Figure 2</b>

**Figure 2

Comparison of the rates of trapping of secretory and standard gene trap vectors. GTSTs matching unique genes (e ≤ 10−6) were identified by using the RefSeq database (rel. 13) and the BlastN algorithm. The number of novel genes hit by accumulating insertions was traced chronologically.

<b>Figure 3</b>

**Figure 3

Correlation between secretory trap insertions and the number of individual genes per chromosome.

<b>Figure 4</b>

**Figure 4

Distribution of gene trap insertions according to the position of the trapped intron within genes. The data are based on NCBI mouse genome build 34 and RefSeq database release 13.

<b>Figure 5</b>

**Figure 5

CD2 expression by U3Ceo trapped ES cells. A total of 5 × 104 cells were infected with U3Ceo retrovirus selected in G418. A total of 1 × 106 NeoR cells (trapped TBV-2) were treated with the Leu-5b monoclonal mouse anti-human CD2 antibody. Similarly treated wild-type (TBV-2) cells served as a negative control. CD2 expression was estimated by flow cytometry after treating the cells with a FITC-conjugated sheep anti-mouse Fab′2 antibody.

<b>Figure 6</b>

**Figure 6

Transcriptional analysis of X-linked mutations in trapped ES cell lines. (A) RT–PCR of fusion transcripts induced by the gene trap insertions using trapped gene- and vector- specific primers. M, molecular weight marker (50 bp ladder). (B) Quantitative RT–PCR of wild-type transcripts expressed in the trapped ES cell lines. Gene-specific primers were chosen in exons flanking the insertion sites (Supplementary Table 1 and Supplementary Figure 1). Results are means from three independent experiments ± SD.

<b>Figure 7</b>

**Figure 7

Secretory gene disruption in transgenic mice. (A) Top: structure of the C030019F02Rik gene as annotated by ENSEMBL and position of the gene trap insertions (arrow). Bottom: northern blot analysis of C030019F02Rik transcripts expressed in homozygous mice. 20 µg total RNAs extracted from the brains of wild type (+/+) and mutant (−/−) mice were fractionated on 1% formaldehyde/agarose gels and hybridized to a 32P-labeled full-length C030019F02Rik c-DNA probe. (B) Top: structure of the semaphorin-4B gene as annotated by ENSEMBL and position of the gene trap insertion (arrow). Bottom: RT–PCR of transcripts expressed in wild type (+/+) and homozygous mutant (−/−) embryos using primers complementary to the 11th and 14th exon of the semaphorin-4B gene. M, molecular weight marker (smart ladder).

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