Site-Specific Recombination with Inverted Target Sites: A Cautionary Tale of Dicentric and Acentric Chromosomes

Abstract

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Keywords: Cre; Drosophila; FLP; FRT; dicentric; lox; mouse; recombinase; sister chromatid; site-specific recombination.

Figure 1

Intramolecular and intermolecular site-specific recombination. Schematic representations of recombination between recombinase targets on the same chromatid or on sister chromatids. (A) Intramolecular recombination between targets in direct orientation results in excision of the material between the targets as an extrachromosomal circle. (B) Intramolecular recombination between inverted repeats inverts the orientation of the DNA between the target sites. (C) Intermolecular recombination between targets at the same location on sister chromatids results in equal exchange. (D) Exchange between targets in direct orientation at different sites on sisters results in duplication and deletion of intervening DNA. (E) Recombination between inverted target sites on sisters produces a dicentric chromosome and an acentric chromosome. Chromatids, are indicated as lines, target sites as arrowheads, and centromeres as filled circles.

Figure 2

Dicentric chromosomes in Drosophila larval neuroblasts. (A) The normal D. melanogaster karyotype. (B) Dicentric and acentric chromosomes formed by unequal sister chromatid exchange using a FlipFlop construct inserted at 37B on chromosome 2. Normal chromosomes are indicated in blue; dicentric (Dc) and acentric (Ac) products indicated in red. (C) Dicentric/acentric chromosomes produced using a FlipFlop construct at 82E on chromosome 3.

Figure 3

Dicentric chromosomes produced in mouse cells. Following exposure to Cre, asymmetric recombination between inverted loxP sites targeted to a locus near (A) the end of chromosome 7, or (B) near the centromere of chromosome 15, would result in a dicentric chromosome and an acentric chromosome fragment. As indicated, the recombinant chromosome products are expected to be significantly different in size.

Figure 4

Evidence of Cre-mediated dicentric and acentric chromosome formation in mouse cells. Pictured here are mitotic figures of cultured cells transfected with a Cre expression vector or purified Cre protein. The targeted chromosomes were visualized with DAPI (A, C, E, and G), or DAPI plus whole chromosome paints (B, D, F, and H) specific for either chromosome 15 (A, B, E, and F) or 7 (C, D, G, and H). Exposure to Cre for a short duration (∼24 hr) results in large acentric fragments (A and B) or short acentric fragments and a large dicentric chromosome (C and D). Longer exposure to Cre results in evidence of further chromosome damage, such as smaller than expected chromosomes (E and F) or extraordinarily long chromosomes (G and H). Arrows indicate acentric fragments and arrowheads indicate dicentric chromosomes.

Figure 5

Dicentric bridge formation in early embryos. Mouse embryos carrying two inverted loxP sites on chromosome 7 with maternal Cre and Histone-GFP were used to make time-lapse movies of the early cleavage divisions. (A and B) are images of two successive timepoints during anaphase showing persistence of a dicentric chromosome during mitotic anaphase (arrow).

Figure 6

Marking cells after loss of acentric chromosome. (A) Schematic representation of the transgene targeted to the tip of chromosome 19. The transgene encodes a minimal CMV promoter (PCMV), the tetracycline operator sequence (tetO7), and the tdTomato coding sequence, centromere proximal to inverted loxP sites; a strong CAG promoter (PCAG) controlling expression of the Tet^R-KRAB repressor gene lies distal to the loxP sites. Loss of the PCAG-tet^R-KRAB portion relieves repression of tdTomato expression, resulting in cells that fluoresce red. Early embryos carrying this construct, and maternally deposited Cre protein, were examined for tdTomato fluorescence. Nuclei are visualized with histone-GFP (green). Cells that have lost the Tet^R gene express tdTomato (red): two cells in (B) and ∼6 cells in (C).