The Different Types of Chromosomal Rearrangements Produced by the Cre/<i>loxP</i> Recombinase System

<div><p>Deletions, duplications, or inversions can be produced depending on the relative orientation of the <i>loxP</i> sites, on their position on the homologous chromosome (i.e., in <i>cis</i> or in <i>trans</i>), and on the cell cycle stage during which the Cre-mediated recombination occurs (G1 or G2).</p><p>(A) Recombination between <i>loxP</i> sites (green arrowhead) integrated in the same orientation in a <i>cis</i> configuration during the G1 phase can only generate a deletion of the region of interest (red arrow); the same configuration in the G2 phase can result in the creation of a deletion and a duplication.</p><p>(B) The deletion and the corresponding duplication can also be obtained from a <i>trans</i> configuration in both G1 and G2 phases. This represents the best configuration to establish the deleted and duplicated chromosomes in the mouse, with both chromosomes compensating for each other with regard to genetic dosage, thus reducing the potential consequence of haploinsufficiency.</p><p>(C and D) When the <i>loxP</i> sites are oriented in opposite directions in a <i>cis</i> configuration, an equilibrium with two forms, inverted and non-inverted, is obtained if the Cre is expressed in G1, while a more likely unstable recombined pair of acentric and dicentric chromosomes is generated if Cre reacts on <i>loxP</i> sites after the S phase or from a <i>trans</i> configuration. From all these recombinant alleles, only those containing the reconstituted mini-gene, however, will be retained during the selection in vitro. Recombinant ES-cell clones should be extensively characterized to verify the engineered chromosome (by Southern blot analysis, normal and quantitative PCR, or FISH).</p><p>Del, deletion; Dup, duplication; Inv, inversion; Rec, one of the original recombinant alleles.</p></div

Robustness of the Tc1 induced phenotypes observed in different genetic backgrounds and interference with different rescues.

<p>(+), (=), (−) or (±) respectively indicate a reported effect with an improvement, similar or with an impairment compared to the control littermates. The (±) corresponds to a partial rescue compared to the transchromosomic Tc1 model. Data adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115302#pone.0115302.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115302#pone.0115302.ref024" target="_blank">24</a>] ^*, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115302#pone.0115302.ref029" target="_blank">29</a>] ^§ and this work.</p><p>Robustness of the Tc1 induced phenotypes observed in different genetic backgrounds and interference with different rescues.</p

Generation and Characterization of Radiation-Induced Deletion Complexes in Mouse ES Cells

<div><p>(A) Insertion of a negative selectable marker (cassette Neo-tk: Hsv-thymidine kinase/neomycin resistance) into a predetermined locus by homologous recombination in F1 hybrid (129/SvJae x C57BL/6J) ES cells (C57BL/6J chromosome represented with a black centromere; 129/SvJae chromosome represented with a white centromere).</p><p>(B) Treatment of the neomycin-resistant targeted cells with radiation to induce the deletions.</p><p>(C) Selection in medium containing 1,2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-5-iodouracil of the colonies having lost the <i>tk</i> gene.</p><p>(D) Characterization of the deletion breakpoints by amplification of the DNA from these clones using primers corresponding to genetic polymorphic markers (represented under the chromosome map by letters a–j) flanking the site of the targeted integration. Deletions are represented as solid boxes.</p></div

Learning and Reversal reference memory performance of mice in the Morris water maze.

<p>(A) The distance traveled on left panel (m) and the percentage of distance traveled in the target quadrant (SE) during the probe trial (S7) on the right (with data from every individual shown), are shown during learning in the Morris water maze. Mice from the four genotypes learned where the platform is located (SE) with a reduction of the distance traveled to find the platform over the 6 learning sessions (S1 to S6). In the right panel, the horizontal line indicates the distance travelled using a random search strategy 25%. No difference was observed for the different genotypes. (B) In the consecutive reversal session we observed similar learning capacities when the platform position was located in the NW quadrant, and after five reversal learning days (RS1 to RS5) shown on the left. During the following probe trial (right panel, RS6), individuals from all the genotypes spent more time in the new target quadrant (NW). Values represent mean + s.e.m.</p

Strategies for In Vivo Cre-Mediated Recombination

<div><p>(A) The general principle of TAMERE is based on two successive breedings in order to have in one male, named the trans-loxer, the <i>Sycp1Cre</i> (<i>Synaptonemal Complex protein 1</i>) transgene and the two <i>loxP</i> sites in a <i>trans</i> configuration, inserted previously in the same orientation at each targeted locus, that define the genetic interval. The <i>Synaptonemal Complex protein 1</i> promoter drives Cre expression at prophase of meiosis in male spermatocytes when chromatid pairs are closely aligned, in order to facilitate the chromatid exchange, leading to the formation of the deletion and the duplication of the interval delimited by the two <i>loxP</i> sites. The last step consists in mating trans-loxer males with wild-type females to generate, in the progeny, individuals carrying the deletion or the duplication of the targeted region.</p><p>(B) The STRING approach takes advantage of a classical crossing-over to bring the two <i>loxP</i> sites into a <i>cis</i> configuration to generate a deletion. Two parental mice (F₀) carrying <i>loxP</i> sites flanking a selected region are crossed. The F₁ progeny containing the two <i>loxP</i> sites are then mated to wild-type mice. The offspring are screened for meiotic crossing-over between both sites leading to mice carrying the <i>loxP</i> sites in a <i>cis</i> configuration. In the subsequent cross, a ubiquitously expressed Cre transgene is introduced, generating the deletion that is established in the next F₄ generation.</p><p>Del, deletion; Dup, duplication.</p><p>red arrow, region of interest; green arrow, <i>loxP</i> site.</p></div

Open field locomotor activity of mice.

<p>The distance travelled (m) during the exploration of the open-field by mice with different genotypes are shown Tc1/Ms2Yah during the consecutive 0–10, 10–20, 20–30 min intervals. The activity of Tc1/Ms2Yah was increased mainly during the first 10 minutes (parameter “distance travelled”, Two-way ANOVA “0–10 min”, F(2,111) = 12.524 p < 0.001; Tuckey’s post hoc method “wt vs Tc1/Ms2Yah” q = 4.663, p = 0.007; “Ms2Yah vs Tc1/Ms2Yah” q = 5.108, p = 0.003 and “Tc1 vs Tc1/Ms2Yah” q = 3.968, p = 0.03Tc1/Ms2YahTc1/Ms2Yah). Values are means + s.e.m.</p

Effect of the sodium channel blocker flecainide on the duration (ms) of P, R, and QRS waves and RR, PR, QT, and QTc intervals in Ts65Dn mice (N = 8).

<p>Significance is given between control and flecainide with <i>P</i> values of 0.05 (*). 0.01 (**) and 0.001 (***).</p

Ratio of the expression level of 5 genes located on Mmu16 in the heart of mutant compared to disomic control mice.

<p>The analysis was done by qRT-PCR. Significance is given with <i>P</i> values of 0.05 (*). 0.01 (**).</p

Genes deregulated in the heart of Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah compound mice.

<p>Only the genes for which the Anova test is below 0.1 and which are not triplicated on the Ts65Dn minichromosome are listed here. (<i>P</i> values: ***: <i>P</i><0.001; **: <i>P</i><0.01; *: <i>P</i><0.05).</p

ECG analyses of urethane anaesthetized Ts65Dn adult mice revealed cardiac conduction anomalies.

<p>Peripheral (A) and precordial (B) leads are presented for the wt and the Ts65Dn mice in the left and right columns respectively. Wt ECG showed a smooth tracing in all leads. P wave, QRS complex and J wave are clearly distinguishable. Ts65Dn ECG showed notching of the S wave in most leads. In V1, QRS amplitude was reduced with a fragmented RSR'S' complex. A slurr was found in the Vms lead. Notice the prolonged PR interval. Representation of Vms lead from wt (C) and Ts65Dn (D) showed measurements performed on wave amplitudes and durations. The isoelectric line (zero) was taken as the mean potential just preceding the R wave and J and T waves were labeled. The T wave was always positive in this lead while it could be elusive in other leads. ECG features (E) were classified according to the presence of fragmented QRS (RSR'S'), slurr and S wave absence (Vx>0) in the precordial leads. 62% of the Ts65Dn mice showed 3 anomalies while 61% of the wt was free of such features. Frontal QRS electrical axis distribution (F) was evenly distributed in Ts65Dn while that of wt animals showed a leftward preferential orientation. Measurement of wave amplitudes in precordial leads (G) showed a large decrease of S and J waves on V1 and limited changes in V4 whereas R waves were found normal in both V1 and V4 (n = 31 and 29 for wt and Ts65Dn respectively) (Student t-test p-values: **: <i>P</i><0.01; ***: <i><u>P</u></i><0.001).</p