<i>Ppd</i> characteristic caudal mass/ectopic limb phenotypes in engineered offspring.

<p>A. (one panel) Bruce4.G9 targeted ES cell clone (9281)-derived chimeric male produced a female heterozygous carrier (Neo⁺/eETn⁺) that was mated to a β-actin FLPe male to produce the newborn (Neo⁻/eETn⁺) shown in the panel here. Genotyping confirmed the proper FLPe recombination fragment demonstrating Neo cassette removal in this mouse. B., C. Typical caudal masses with ectopic limbs in offspring of Neo⁻/eETn⁺ mice. 9 out of 69 offspring from mating between Neo⁻/eETn⁺ males X B6/D2 F₁ females demonstrated typical caudal masses/ectopic limbs (3 panels in B), whereas 8 out of 31 offspring from mating between Neo⁻/eETn⁺ males X FVB females demonstrated typical caudal masses and in one mouse a bifurcated tail seen at birth and later at 3 weeks (5 panels in C).</p

Variation in occurrence of malformations at birth is not correlated with 5′ LTR promoter methylation.

<p>Independent clones generated by PCR were sequenced and analyzed for CpG methylation at 8 sites (7 LTR and 1 X chromosome genomic) upstream of the 5′ LTR in 2 <i>Ppd</i> carrier females with normal phenotypes (NL-Ppd) versus 2 <i>Ppd</i> carrier females with abnormal phenotypes (A-Ppd). The number of clones for each sample with the indicated number of methylated CpGs are tabulated. No difference was observed at these CpG sites either in number or distribution of sites of methylation between these groups. For comparison, CpG methylation was also assessed in affected and unaffected <i>Ppd</i> males and in ES cell line Ppd-D5. In this ES cell line the LTR is almost completely unmethylated, whereas in postnatal adult males the ETn LTR is methylated to a variable extent, but less than in females. cm = caudal mass tissue of mouse #3412.</p

Expected Mendelian numbers of <i>Ppd</i> X-chromosomes at E9.5.

<p>Expected Mendelian numbers of <i>Ppd</i> X-chromosomes at E9.5.</p

Reduced <i>Ppd</i> chromosomes at birth – CZECHII/EiJ cross.

<p>Reduced <i>Ppd</i> chromosomes at birth – CZECHII/EiJ cross.</p

Typical <i>Ppd</i> caudal duplications, among numerous anomalies observed in mutants.

<p>Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967-Lehoczky1" target="_blank">[15]</a> for a complete description of other anomalies, listed in the Introduction. Variation in presentation of ectopic legs with or without caudal masses has been observed.</p

<i>Dusp9</i> mRNA and protein are increased in mutant ES cells.

<p>A, B. Taqman confirmation of increased <i>Dusp9</i> mRNA in mutant ES cells. ES cell steady-state <i>Dusp9</i> mRNA quantitation in ES cell lines (D3, C4, and D5) derived from the original <i>Ppd</i> strain (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#s5" target="_blank">Materials and Methods</a>) compared with Bruce4.G9 ES cell RNA. Steady-state <i>Dusp9</i> mRNA levels in engineered eETn targeted ES cell lines (928B, 928D, 9281, 9284, 9287) derived by homologous recombination in Bruce4.G9 cells, compared to <i>Dusp9</i> expression in wild-type Bruce4 ES cells. All targeted ES cell lines also demonstrate a large increase in <i>Dusp9</i> mRNA compared to Bruce4.G9; n = 3 independent RNA preparations. Note variability of RNA expression across different ES cell lines. C. Increased expression of DUSP9 protein in mutant ES cells. DUSP9 protein was quantitated by Western blot with antibody to DUSP9 (top blot), produced and kindly supplied by R. Dickinson, relative to β-actin (lower blot) from normal ES cells (PAT-5, ND-D3, UMB6J-D7, and Bruce4.G9) compared to original <i>Ppd</i> mutant-derived ES cells (Ppd-D3, Ppd-D5, Ppd-C4) and eETn ES cells (as shown on right, blue bars); graph below represents data from two separate protein extraction experiments. Bruce4.G9 is the parent ES cell line for eETn cells. Data with the second antibody (anti-MKP4; Santa Cruz Biotech.) was identical (data not shown). Pre-incubation of each antibody with a synthesized peptide to DUSP9 reduced or eliminated the signal observed in ES cells, and pre-incubation with a control peptide did not visibly alter the DUSP9 signals (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967.s005" target="_blank">Figure S5</a>).</p

Reduced <i>Ppd</i> chromosomes at birth – C3H cross.

<p>Reduced <i>Ppd</i> chromosomes at birth – C3H cross.</p

Schematic diagram of the targeting vector strategy and expected results of homologous recombination and Flp-mediated selection cassette removal.

<p>Ppd BAC clone 2 was the starting material from which a 5′ homology segment of 5 kb beginning in <i>Dusp9</i> intron 1 at GRCm38 position ChrX: 73639873 including exons 2, 3, and 4, as well as a 3′ homology arm extending 10 kb toward <i>Pnck</i> including the ETn and ending at GRCm38 position ChrX: 73655955, was excised from the BAC and used for recombineering as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#s5" target="_blank">Materials and Methods</a>. ES cell clone DNA was subjected to Southern blotting with Probe A (located outside of the 5′ homology arm) after XmnI digestion (labeled as X restriction site in the figure; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967.s002" target="_blank">Figure S2</a>), as well as PCR with F5 and R6, to identify successful homologous recombinants. FLP-mediated recombination between the two FRT sites (blue triangles) and removal of the PGK-Neo cassette was accomplished by mating Neo⁺/eETn⁺ females with β-actin FLPe male mice obtained from Jackson Labs. Successful FLP-mediated recombination was verified in offspring by PCR/sequencing with X chromosome specific primer P₁ (5′-CAAATGCCTGAGCTGATAAAATAA-3′) and LTR specific primer P₂ (5′-CCCTTCCTTCATAACTGGTGTC-3′) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003967#pgen.1003967.s003" target="_blank">Figure S3</a>).</p

Location and orientation of new ETnII-β insertion in the <i>Ppd</i> genetic interval relative to immediately adjacent genes.

<p>The ETn insertion site is located between <i>Dusp9</i> and <i>Pnck</i>, ∼1.6 kb downstream and antisense to <i>Dusp9</i>. Transcription proceeds from the 5′ LTR toward <i>Dusp9</i>. PCR primers used for genotyping mutants (F5/R6) are shown.</p