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

<i>Lago1</i> gene targeting by HR at the <i>Dct</i> locus.

<p>(A) Insertion of <i>Lago1</i> gene at the <i>Dct</i> locus. The <i>Dct^I-SceI</i> allele, the HR1 repair vector and the <i>Dct^Lago1</i>^-<i>Neo</i> targeted allele are represented from top to bottom. 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> and <i>Dct^Lago1</i>^{-<i>Neo/+</i>} ES cells. Genomic DNAs were digested with <i>Bam</i>HI. The probe used for the hybridization is the external 5′ probe depicted by a black bar. The 11.7, 4.5, and 10.1 kb fragments are distinctive of the <i>Dct⁺</i>, <i>Dct^I-SceI</i>, and <i>Dct^Lago1</i>^-<i>Neo</i> alleles, respectively. (C) Diagram of DSB-induced homologous recombination with no insertion of the <i>Lago1</i> gene. The <i>Dct^I-SceI</i> allele, and the HR1 repair vector are represented from top to bottom. The short regions of homology, 58 bp and 125 bp in length, between the HR1 repair vector and the genomic DNA at the <i>Dct</i> locus in <i>Dct^I-SceI/+</i> cells, are depicted by grey rectangles. An HR between these two short regions of homology would lead to loss of the I-<i>Sce</i>I site with no integration of the Neo^R cassette. The resulting cells would die in the presence of G418.</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

Construction of the HR1 repair vector.

<p>The pENTR1ApA entry vector is represented in the upper left. Black circles flanking <i>ccdB</i> represent multi-cloning sites (MCSs). The SV40 polyadenylation sequence (pA) was introduced at the 3′ end of the <i>ccdB</i> gene, before <i>att</i>L2 sequence. The entry vector contains a Kan^R cassette conferring resistance to kanamycin in <i>E. coli</i>. The entry vector carrying <i>Lago1</i> gene is represented in the upper right. The destination vector (DV1) contains 1.4 and 4.5 kb of <i>Dct</i> homologous arms depicted as grey rectangles. DV1 also contains the Neo^R and HSV-TK cassettes used in cell culture, and an Amp^R cassette conferring resistance to ampicillin in <i>E. coli</i>. The repair vector (HR1) is produced by LR reaction, allowing the replacement of Cm^R-<i>ccdB</i> cassette by the <i>Lago1</i> gene.</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