Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization

Abstract

One of the challenges of genomic research after the completion of the human genome project is to assign a function to all the genes and to understand their interactions and organizations. Among the various techniques, the emergence of chromosome engineering tools with the aim to manipulate large genomic regions in the mouse model offers a powerful way to accelerate the discovery of gene functions and provides more mouse models to study normal and pathological developmental processes associated with aneuploidy. The combination of gene targeting in ES cells, recombinase technology, and other techniques makes it possible to generate new chromosomes carrying specific and defined deletions, duplications, inversions, and translocations that are accelerating functional analysis. This review presents the current status of chromosome engineering techniques and discusses the different applications as well as the implication of these new techniques in future research to better understand the function of chromosomal organization and structures.

Figure 1. Generation and Characterization of Radiation-Induced Deletion Complexes in Mouse ES Cells

(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). (B) Treatment of the neomycin-resistant targeted cells with radiation to induce the deletions. (C) Selection in medium containing 1,2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-5-iodouracil of the colonies having lost the tk gene. (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.

Figure 2. The Different Types of Chromosomal Rearrangements Produced by the Cre/loxP Recombinase System

Deletions, duplications, or inversions can be produced depending on the relative orientation of the loxP sites, on their position on the homologous chromosome (i.e., in cis or in trans), and on the cell cycle stage during which the Cre-mediated recombination occurs (G1 or G2). (A) Recombination between loxP sites (green arrowhead) integrated in the same orientation in a cis 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. (B) The deletion and the corresponding duplication can also be obtained from a trans 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. (C and D) When the loxP sites are oriented in opposite directions in a cis 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 loxP sites after the S phase or from a trans 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). Del, deletion; Dup, duplication; Inv, inversion; Rec, one of the original recombinant alleles.

Figure 3. Strategies for In Vivo Cre-Mediated Recombination

(A) The general principle of TAMERE is based on two successive breedings in order to have in one male, named the trans-loxer, the Sycp1Cre (Synaptonemal Complex protein 1) transgene and the two loxP sites in a trans configuration, inserted previously in the same orientation at each targeted locus, that define the genetic interval. The Synaptonemal Complex protein 1 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 loxP 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. (B) The STRING approach takes advantage of a classical crossing-over to bring the two loxP sites into a cis configuration to generate a deletion. Two parental mice (F₀) carrying loxP sites flanking a selected region are crossed. The F₁ progeny containing the two loxP sites are then mated to wild-type mice. The offspring are screened for meiotic crossing-over between both sites leading to mice carrying the loxP sites in a cis configuration. In the subsequent cross, a ubiquitously expressed Cre transgene is introduced, generating the deletion that is established in the next F₄ generation. Del, deletion; Dup, duplication. red arrow, region of interest; green arrow, loxP site.