Mendelian inheritance in the offspring of <i>Btg1^-</i>, and <i>Btg2</i> single knockout mice.

<p>Expected (exp.) mendelian inheritance and observed (obs.) inheritance after WT, <i>Btg1</i> and <i>Btg2</i> breedings.</p><p>P-values of the mendelian ratios are calculated with the Chi-square test and are either significant if p <0.05 or not significant (n.s).</p><p>Mendelian inheritance in the offspring of <i>Btg1^-</i>, and <i>Btg2</i> single knockout mice.</p

Overview of the skeletal phenotypes in <i>Btg1-</i> and <i>Btg2</i>-deficient mice.

<p>Axial vertebrae are indicated by different colors: green (cervical), yellow (thoracic), blue (lumbar) and red (sacral). The thoracic ribs are indicated by the grey horizontal lines, of which T1-T7 form sternocostal junctions with the sternum. <i>Btg1-</i> and <i>Btg2-</i>deficient mice display C7 to T1 posterior transformation, while <i>Btg1</i>^-/- mice show also partial or complete L6 to S1. <i>Btg2^-/-</i> mice display in most cases T13 to L1 and complete L6 to S1 transformations. <i>Btg1^-/-</i>,<i>Btg2^-/-</i> double knockout animals display a more pronounced phenotype with C7 to T1, T13 to L1 and L6 to S1 homeotic transformations.</p

Characterization of <i>Btg1^-/-</i>, <i>Btg2^-/-</i> and <i>Btg1^-/-;Btg2^-/-</i> mice.

<p>(A) The mouse <i>Btg1</i> gene is disrupted by insertion of a neomycin resistance cassette via <i>SacII</i> restriction sites in the first exon. The second exon of the <i>Btg2</i> gene is replaced by a neomycin cassette in the antisense direction. The arrows indicate the position of primers used for genotyping. (B) Genotyping of mice was performed by PCR on genomic DNA using primers specific for the Btg1 and Btg2 wild-type (WT) and knockout (KO) allele. (C) Number of pups obtained from wild-type, heterozygous and homozygous <i>Btg1</i>, <i>Btg2</i> and <i>Btg1xBtg2</i> breedings. Data are from at least 4 independent crossings. *, <i>P</i>< 0.05, **, <i>P</i>< 0.01, ***, <i>P</i>< 0.001.</p

Mendelian inheritance in the offspring of <i>Btg1; Btg2</i> double knockout mice.

<p>Expected (exp.) mendelian inheritance and observed (obs.) inheritance after intercrossing compound <i>Btg1;Btg2</i> knockout animals.</p><p>P-values of the mendelian ratios are calculated with the Chi-square test and are either significant if p <0.05 or not significant (n.s).</p><p>Mendelian inheritance in the offspring of <i>Btg1; Btg2</i> double knockout mice.</p

Posterior homeotic transformation of the thirteenth thoracic vertebra in mice deficient for <i>Btg2</i>.

<p>(A-D) Dorsal view of the cervicothoracic region of the skeleton in 18.5 dpc wild-type, <i>Btg1^-/-</i>, <i>Btg2^-/-</i> and <i>Btg1^-/-;Btg2^-/-</i> embryos stained with alizarin red and alcian blue. (A-B) Wild-type <i>C57BL6/J</i> mice have thirteen thoracic ribs, while <i>Btg1</i>-deficient mice display fourteen ribs due to the extra extensive rib at C7. (D) Although the T13 rib is absent in <i>Btg1^-/-;Btg2^-/-</i> mice they still have thirteen thoracic ribs due to the C7 to T1 posterior transformation.</p

<i>Btg1;Btg2</i> double knockout mice display posterior homeotic transformation at the lumbo-sacral transition.

<p>Skeletal defects in 18.5 dpc wild-type, <i>Btg1^-/-</i> and <i>Btg1^-/-;Btg2^-/-</i> embryos. (A-C) Dorsal view showing the lumbar-sacral regions. (B) <i>Btg1^-/-;Btg2^-/-</i> mice frequently show five lumbar vertebrae compared to six in wild-type mice. (C) <i>Btg1^-/-</i> mice may show asymmetric L6 in which the right side (indicated by arrow) indicates a lumbar vertebra and the left side a sacral vertebra.</p

Mendelian inheritance in the offspring of <i>Btg1^-</i>, and <i>Btg2</i> single knockout mice.

<p>Expected (exp.) mendelian inheritance and observed (obs.) inheritance after WT, <i>Btg1</i> and <i>Btg2</i> breedings.</p><p>P-values of the mendelian ratios are calculated with the Chi-square test and are either significant if p <0.05 or not significant (n.s).</p><p>Mendelian inheritance in the offspring of <i>Btg1^-</i>, and <i>Btg2</i> single knockout mice.</p

Targeted deletion of <i>Btg1</i> and <i>Btg2</i> results in malformation of the sternum.

<p>(A-B) Ventral view of the sternum with attached ribs of 18.5 dpc wild-type and <i>Btg1^-/-;Btg2^-/-</i> embryos stained with alizarin red and alcian blue. Wild-type <i>C57BL6/J</i> mice display normal ossification of the five sternbrae, whereas an asymmetric pattern of ossification of the sternebrae is observed in mice lacking both <i>Btg1</i> and Btg2 expression. The xiphoid process (Xip) of the sternum is not affected in <i>Btg1-</i> and <i>Btg2-</i>deficient mice.</p

Skeletal malformations in <i>Btg1</i>- and <i>Btg2</i>-deficient mice.

<p>Skeletal malformations in <i>Btg1</i>- and <i>Btg2</i>-deficient mice.</p

Expression of <i>BTG1</i> truncated read-through transcripts in BCP-ALL cells with <i>BTG1</i> deletions.

<p>(A) Schematic representation of the wild-type human <i>BTG1</i> gene, existing of two partly coding exons, and five different <i>BTG1</i> transcripts due to <i>BTG1</i> gene deletions. Exons are represented by black (coding) or white (non-coding) bars. Indicated are the RT-PCR primers that were used to detect expression of the wild-type <i>BTG1</i> transcript (primers A and B), or one of the <i>BTG1</i> truncated read-through transcripts for deletion II (primers A and C), deletion III (pimers A and D), deletion IV (primers A and E), deletion V (primers A and F), or deletion VIII (primers A and G). (B) RT-PCR analyses on total RNA isolated from the BCP-ALL cell lines Nalm6 and RS4;11 (<i>BTG1</i> wild-type) and REH, SUP-B15 and 380, each with distinct monoallelic <i>BTG1</i> deletions. (C) RT-PCR analyses on primary BCP-ALL samples in which a single <i>BTG1</i> deletion (Pt1, Pt2, Pt3 and Pt5), multiple <i>BTG1</i> deletions (Pt4 and Pt6) or no <i>BTG1</i> deletions were detected with genomic PCR (Pt7). Type of deletions (III, V, or VIII) and outcome of MLPA (p: deletion-positive; n: deletion-negative) are indicated. <i>BTG1</i> read-through transcripts were verified by sequencing (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002533#pgen.1002533.s007" target="_blank">Table S5</a>), except for Pt3-deletion V, which was an unrelated DNA sequence. (D) Quantitative real-time RT-PCR data representing relative expression levels of <i>BTG1</i> measured 5′ (primers exon 1/2) and 3′ of the <i>BTG1</i> breakpoint hotspot (primers exon 2). Expression levels were normalized to <i>HPRT</i> levels, and compared to the expression level in Nalm6, which was set to 1. The data shown represent the average of two independent cDNA reactions and triplicate qRT-PCR reactions.</p