Mouse chromosome engineering for modeling human disease

Abstract

Chromosomal rearrangements are frequently in humans and can be disease-associated or phenotypically neutral. Recent technological advances have led to the discovery of copy-number changes previously undetected by cytogenetic techniques. To understand the genetic consequences of such genomic changes, these mutations need to be modeled in experimentally tractable systems. The mouse is an excellent organism for this analysis because of its biological and genetic similarity to humans, and the ease with which its genome can be manipulated. Through chromosome engineering, defined rearrangements can be introduced into the mouse genome. The resulting mouse models are leading to a better understanding of the molecular and cellular basis of dosage alterations in human disease phenotypes, in turn opening new diagnostic and therapeutic opportunities.

Figure 1

Examples of common chromosomal rearrangements found in humans.

Figure 2

Recombinase reactions catalyzed by Cre. Box: The target site recognized by the P1 bacteriophage recombinase, Cre, is called loxP [locus of cross over (x) in P1]. The red arrow in the core region of the loxP site indicates the direction (orientation) of the loxP site. (a) A cis recombination event between two loxP sites (black triangles) in the same orientation will lead to deletion (Df) or duplication (Dp) of the flanked DNA sequence. (b)If the loxP sites are orientated in opposite directions, the loxP-flanking sequence will be inverted. (c) Recombination between two loxP sites in trans will lead to the reciprocal exchange (translocation) of the regions that flank the loxP sites.

Figure 3

Gene targeting in embryonic stem (ES) cells to enable chromosomal engineering. Insertional targeting vectors, as shown, can be used to insert loxP sites (black triangles), positive selectable markers (such as the neomycin resistance cassette, N, and the puromycin resistance cassette, P), and Hprt gene fragments (5′ and 3′) into the desired endpoints (loci) of ES cells. Expression of the neomycin and puromycin resistance genes allows different targeting events to be selected. The complementary, but nonfunctional, 5′ Hprt and 3′ Hprt fragments are derived from the Hprt minigene and recombination between the two loxP sites will result in union of the two Hprt fragments to generate a functional Hprt minigene, which confers resistance in Hprt-deficient ES cells to the drug HAT (hypoxanthine, aminopterin, and thymidine), thereby enabling in vitro selection of ES cells carrying the desired chromosomal rearrangement (87).

Figure 4

A general strategy for chromosomal engineering in mice. (a) A loxP site is inserted into the first endpoint using gene targeting in embryonic stem (ES) cells (involving recombination between the exogenous targeting vector and the endogenous homologous genomic locus). The targeting vector carries a positive selectable marker gene. (b) A second loxP site, linked to a different positive selectable marker gene, is targeted to the second endpoint (either on the same or a different chromosome). This can be achieved either by gene targeting or random insertion (see Figure 5). (c) Double-targeted ES cell clones, identified by molecular techniques such as Southern blotting, are then exposed to Cre, which catalyzes the recombination between the loxP sites. (d) ES cell clones carrying the desired chromosomal rearrangements are identified using molecular techniques such as Southern blotting, fluorescence in situ hybridization (FISH), and/or comparative genomic hybridization (CGH). (e) These ES cell clones are subsequently injected into mouse blastocysts and the embryos transferred into the uteri of pseudopregnant foster mothers. (f) Chimeras that are generated from the blastocyst injection are then mated with wild-type mice to establish germline transmission of the modified allele. (g) The progeny from these matings are molecularly characterized and a mutant mouse line carrying the engineered chromosome(s) is established.

Figure 5

Nested chromosomal deletions induced with a retroviral vector. The first deletion endpoint is fixed by targeting the 5′ Hprt cassette and a loxP site (black arrow) to a predetermined locus. The 3′ Hprt cassette and a second loxP site are then randomly integrated into the genome of the embryonic stem (ES) cell using a recombinant retroviral vector. Cre catalyzes recombination between the loxP sites and ES cells carrying the deleted chromosomes can be identified (112).

Figure 6

Diagram of the mouse chromosome 16 deletion mutants made by different groups. (a) Df1/Dp1 (60), (b) Lgdel (69), (c) (47), and (d) (85). The red bar indicates the gene content of the transgene that rescues cardiovascular defects in Df1/+ and Lgdel/+ mutants. Arvcf, armadillo repeat gene deleted in velo-cardio-facial syndrome; Comt, catechol-O-methyltransferase; Dgcr2, DiGeorge syndrome gene c; Es2el, expressed sequence 2 embryonic lethal; Gscl, goosecoid-like; Hira, histone cell-cycle-regulation defective homolog A; Pnutl1, peanut-like 1; Ranbp1, Ran-binding protein 1; Slc25a1, solute carrier family 25 member 1; Tbx1, T-box 1; Ufd1l, ubiquitin fusion degradation 1-like; Znf 74, zinc-finger protein 74.

Figure 7

Triplicated regions in trisomy 16 (TS16) mice. (a) TS16 (33), (b) Ts65Dn (24, 89), (c) Ts1Cje (97), (d) Ms1Ts65 (79, 98), and (e) Ts1Rhr (80). In TS16 mice, the whole chromosome 16 is triplicated and the syntenic regions to human chromosomes are indicated. For Ts65Dn, Ts1Cje, Ms1Ts65, and Ts1Rhr mice, the segmental triplicated region is shown and the genes involved are as indicated (the triplicated region in Ts1Cje spans from Sod1 to Mx1; however, Ts1Cje is not functionally triplicated for Sod1 because the Sod1 gene in the translocated segment has been inactivated by the targeting event). App, amyloid ß (A4) precursor protein; Cbr1, carbonyl reductase 1; Grik1, glutamate receptor, ionotropic, kainate1; Sod1, superoxide dismutase 1; Gart, phosphoribosylglycinamide formyltransferase; Dryk1, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a; Mx1, myxovirus (influenza virus) resistence-1; Mx2, myxovirus (influenza virus) resistence-2.

Figure 8

Mouse models of the reciprocal translocations involving the MLL. (a) Example of a knock-in approach: To generate Mll-Af 9 fusion gene mice, a targeting vector consisting of the 3′ terminal end of human AF9 gene (yellow) fused within exon 8 of mouse Mll gene [at the position of fusions found after t(9;11)(p22;q23) is generated]. This targeting vector is introduced into the mouse genome using gene targeting in embryonic stem (ES) cells and the resulting allele transmitted through the germline. Using this strategy, the fusion protein is expressed at all developmental stages and in all tissues, and chimeric mice carrying the fusion protein develop acute myeloid leukemia. (b) Example of a translocator approach: To generate de novo chromosomal translocations in mice, loxP sites are introduced into the relevant genes using gene targeting in ES cells, and chimeric mice carrying the modified alleles are derived. These mice are bred to obtain germline transmission and subsequently interbred with each other before being bred with Cre deleter mice (mice expressing Cre under the control of a specific promoter). Therefore, recombination between the loxP sites and subsequent generation of the fusion gene only occurs in tissues/cell lineages where Cre is expressed (as detailed in the table).