Histological analysis of somitic regions of control and <i>Nle1^mcKO</i> mutant embryos.

<p>Embryos were sectioned and stained with hematoxylin and eosin at E9.5 (transverse section of the tail, <b>A</b>) and at E11.5 (sagittal section of interlimb region, <b>B</b>). Arrow indicated the picnotic nuclei visible in the neural tube of E9.5 <i>Nle1^mcKO</i> embryos. At E11.5, absence of sclerotomal condensations (arrowhead in control) and fusion of dorsal spinal ganglia (sg) are shown.</p

Increased cell death is observed in <i>Nle1^mcKO</i> mutant embryos.

<p><b>A.</b> E9.5 and E10.5 control and mutant embryos were incubated in LysoTracker Red solution. Normal developmental apoptosis was observed in the head (E9.5 and E10.5) and in the ventral part of the somites (E10.5) of control embryos. In <i>Nle1^mcKO</i> embryos, apoptosis in the head was similar as control embryos, whereas a marked increase in apoptosis was observed in the neural tube and the entire somites at E9.5 (n = 2 mutants and n = 6 controls including 2 <i>Meox2^Cre</i> embryos) and E10.5 (n = 3 mutants and n = 8 controls including 2 <i>Meox2^Cre</i> embryos). <b>B.</b> Immunostaining for the active form of caspase3 protein in E9.5 control and <i>Nle1^mcKO</i> embryos. Representative results of immunostaining of transverse section of the caudal part of embryos are shown. In <i>Nle1^mcKO</i> embryos, high number of apoptotic cells was observed (arrowheads) in the neural tube and the somites. (n = 3 mutants and n = 4 controls including a <i>Meox2^Cre</i> embryo). No increase in apoptosis was observed due to the expression of the <i>Meox2^Cre</i> allele.</p

Expression pattern of markers for somitic lineages in E10.5 control and <i>Nle1^mcKO</i> embryos.

<p>Whole-mount <i>in situ</i> hybridizations were performed with <i>Pax1</i>, <i>Paraxis</i>, <i>Pax3</i> and <i>Myf5</i> riboprobes (n = 3 control, n = 3 <i>Nle1^mcKO</i> mutant embryos for <i>Pax1</i>, <i>Paraxis</i> and <i>Pax3</i>, n = 2 control and n = 2 control, n = 2 <i>Nle1^mcKO</i> mutant embryos for <i>Myf5</i>. Pax3 is expressed in the dorsomedial (white arrowhead) and dorsolateral (red arrowhead) lips of the dermomyotome in E10.5 control embryos. The red line indicate the limb bud where <i>Pax3</i>-positive muscle progenitors are visible in the control embryo but not in the <i>Nle1^mcKO</i> embryo. Red boxes mark an embryonic region shown enlarged below each embryo.</p

Number of mutant embryos and pups obtained from crosses between <i>Nle1^flox/flox</i> and <i>Nle1^{LacZ +/}; Meox2^Cre/+</i> mice at various stages.

a<p>Absolute number and frequency (%).</p>b<p>No mutant pups were found alive at birth.</p

Partial recombination of the <i>Nle1^flox</i> allele by <i>Meox2^Cre</i> and detection of <i>Nle1</i>-deficient cells.

<p><b>A.</b> At gastrulation, <i>Meox2^Cre</i>-mediated recombination was detected by staining for β-galactosidase activity using <i>Rosa26^STOP-LacZ</i> reporter allele in <i>Rosa26^STOP-LacZ/+; Nle1^flox/+; Meox2^Cre/+</i> control embryo (left, n = 3) and in <i>Rosa26^STOP-LacZ/+; Nle1^flox/Δ; Meox2^Cre/+</i> mutant embryo (right, n = 3). <b>B.</b> PCR amplification of genomic DNA from <i>Nle1^flox/+; Meox2^Cre/+</i> control embryos showing partial recombination of the <i>Nle1^flox</i> allele at E7.5 (n = 4), E8.5 (n = 12). The recombined (<i>Nle1</i>^Δ) allele is detected as soon as E7.5. Note that PCR analysis underestimates the recombination efficiency at this stage. Indeed, PCR analysis was performed on whole embryo containing extraembryonic tissues (visceral endoderm, extraembryonic ectoderm) that do not express the Cre recombinase. Presence of <i>Nle1</i>-deficient cells was evaluated by PCR amplification of genomic DNA performed on <i>Nle1^mcKO</i> mutant embryos at E7.5 (n = 9) and E8.5 (n = 6). Representative results of PCR are shown. <b>C.</b> PCR amplification of genomic DNA from <i>Nle1^flox/+; Meox2^Cre/+</i> control embryos showing partial recombination of the <i>Nle1^flox</i> allele in several tissues at E10.5 (n = 3) and E15.5 (n = 3). Efficiency of recombination was estimated through comparison with a reference DNA sample (*) derived from a <i>Nle1^flox/</i>^Δ adult mouse. Nested PCR were performed for the E7.5 and E8.5 stages. sp. cord: spinal cord, ag: adrenal glands.</p

Frequency of homologous recombination at the <i>Dct</i> locus.

<p>Frequency of homologous recombination at the <i>Dct</i> locus.</p

H₂B-<i>mCherry</i> gene targeting by HR at the <i>Dct</i> locus.

<p>(A) Insertion of H₂B-<i>mCherry</i> gene at the <i>Dct</i> locus. The <i>Dct^I-SceI</i> allele, the HR2 repair vector and the <i>Dct^H2B</i>^{-<i>mCherry</i>-<i>Neo</i>} allele are represented from top to bottom. <i>Avr</i>II sites are indicated. There are no homologous sequences between <i>Dct^I-SceI</i> and HR2 close to the I-<i>Sce</i>I site. A lightning denotes I-<i>Sce</i>I expression from pCMV-I-<i>Sce</i>I or pCAG-I-<i>Sce</i>I plasmid. The 1.4 and 4.5 kb of <i>Dct</i> isogenic DNA are depicted by grey rectangles. (B) Southern blot analysis of <i>Dct^+/+</i>, <i>Dct ^I-SceI/+</i>ES cells, and <i>Dct^{H2B-mCherry-Neo/+}</i> ES clones 7E and 11E. ES clones 7E and 11E were obtained after transfection with the linear HR2 repair vector, and with both the supercoiled HR2 repair vector and pCAG-I-SceI expression plasmid, respectively. Genomic DNAs were digested with <i>Avr</i>II. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 3.9 kb fragment is carried by the <i>Dct ⁺</i> and <i>Dct ^I-SceI</i> alleles, and the 6.5 kb fragment is distinctive of the <i>Dct ^{H2B-mCherry-Neo}</i> targeted allele.</p

Assay of double-strand break induced by I-<i>Sce</i>I at the <i>Dct</i> locus.

<p>(A) Diagram of the ligation-mediated PCR (LM-PCR) technique to analyze a lesion at the I-<i>Sce</i>I site. The successive steps of LM-PCR are represented from top to bottom. Arrows indicate the <i>Dct</i> gene-specific primers LM-C1, LM-C2 and LM-C3. Two black bars depict the asymmetrical synthetic double-stranded linker constituted of linkerF and linkerR primers. After cleavage and denaturation of genomic DNA, LM-C1 primer was annealed and extended. Then, the double-stranded linker was ligated to the blunt-ended fragment. Fragments were PCR amplified using LM-C2 and linkerF primers. A second nested PCR amplification was performed using LM-C3 and linkerF primers. A lesion at the I-<i>Sce</i>I site would lead to a 148 bp LM-PCR product. (B) Detection of cleavage by the LM-PCR assay. Genomic DNA from mock-transfected MF1 ES cells was extracted, treated <i>in vitro</i> with <i>Pst</i>I (lane 1) or I-<i>Sce</i>I (lane 2), and analyzed by LM-PCR. After an autoradiographic exposure time of 2 h, both long and short PCR products (200 bp and 148 bp) are seen, as expected for <i>Pst</i>I and I-<i>Sce</i>I-treated DNA. The positions of size standards (in bp) are shown on the left. (C) Induction of DSBs by I-<i>Sce</i>I in ES cells. Genomic DNA from mock-transfected, pCMV-I-<i>Sce</i>I and pCAG-I-<i>Sce</i>I expression plasmid-transfected MF1 ES cells was extracted and analyzed by LM-PCR. After an autoradiographic exposure time of 16 h, no LM-PCR products is observed when DNA from mock-transfected cells was used as a template. A fragment of the predicted 148 bp size is seen after LM-PCR-amplification of DNA from cells transfected with pCMV-I-<i>Sce</i>I and pCAG-I-<i>Sce</i>I. The positions of size standards (in bp) are shown on the left.</p

Production of a new target allele at the <i>Dct</i> locus.

<p>(A) Introduction of an I-<i>Sce</i>I site at the <i>Dct</i> locus. From top to bottom are represented the <i>Dct</i> wild-type allele (<i>Dct⁺</i>), the replacement vector, the <i>Dct^I-SceI-Neo</i> targeted allele, and the <i>Dct^I-SceI</i> allele produced after deletion of the Neo^R cassette. The grey boxes represent exons 1 and 2 of the <i>Dct</i> gene. The black circle represents 109 bp of <i>Dct</i> intron 1 sequence that are lost during an homologous recombination event. The horizontal black bar represents the external 5′ probe used for the Southern blots. The Neo^R and HSV-TK cassettes are depicted as white rectangles. <i>loxP</i> sites are represented by white triangles. The <i>Dct</i> homologous arms, 1.9 and 4.5 kb in length, are denoted as grey rectangles. I-<i>Sce</i>I and <i>BamH</i>I restriction sites are indicated. (B) Southern blot analysis of <i>Dct^+/+</i> ES cells and targeted ES cells (clone 4). Genomic DNAs of ES cells were digested with <i>Bam</i>HI. The 11.7 and 6.4 kb fragments are distinctive of the <i>Dct⁺</i> and <i>Dct^I-SceI-Neo</i> alleles, respectively. (C) Test of the ability of I-<i>Sce</i>I meganuclease to specifically cleave <i>Dct^I-SceI-Neo</i>^/+ ES cells. Southern blot analysis of <i>Dct^+/+</i> ES cells and clone 4. Genomic DNAs were digested with I-<i>Sce</i>I and <i>Bam</i>HI. The 4.5 kb fragment is distinctive of the <i>Dct^I-SceI-Neo</i> allele. (D) Southern blot analysis of <i>Dct^+/+</i> ES cells, clone 4, MF1 and MF2 clones. Genomic DNAs were digested with <i>Bam</i>HI. The 4.5 kb fragment is distinctive of the <i>Dct^I-SceI</i> allele.</p

Construction of the HR2 repair vector.

<p>The pENTR1ApA entry vector is represented in the upper left. The entry vector containing the H₂B-<i>mCherry</i> gene is represented in the upper right. The DV2 destination vector contains the <i>Dct</i> homologous arms, 1.4 and 4.5 kb in length, depicted as grey rectangles. The black circle denotes 109 bp of <i>Dct</i> intron absent in DV1 destination vector that were inserted in DV2 destination vector. DV2 also contains a Neo^R cassette flanked with <i>FRT</i> sites depicted as white diamond symbols. The repair vector (HR2) is produced by LR reaction, allowing the replacement of Cm^R-<i>ccdB</i> cassette by the H₂B-<i>mCherry</i> gene.</p