Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids

Abstract

The bacteriophage P1 Cre͞loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic ''pseudo-loxP'' sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre͞loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.T he bacteriophage P1 recombinase, Cre, is a member of the integrase family of site-specific recombinases that catalyzes recombination between loxP DNA elements as a part of the normal viral life cycle (1, 2). All members of the integrase family share a similar mechanism of action (3). In Cre-mediated recombination, individual loxP sites are bound by Cre homodimers (4). Two Cre-bound loxP sites interact in the recombination reaction. The loxP site is a 34-bp element consisting of two 13-bp inverted repeats separated by a directional 8-bp core (1, 5-7). During cleavage, Cre Tyr 324 becomes covalently linked to the 3Ј-phosphate, forming a DNA-phosphotyrosine intermediate (8, 9). Interaction with a second loxP-bound Cre dimer allows nucleophilic attack of the 3Ј-phosphotyrosine in one loxP site by the free 5Ј-hydroxyl of the other. This ligation reaction induces a structural rearrangement of the tetrameric complex, allowing the same reaction to occur on the second strand of each loxP site, thus completing the strand exchange reaction (8,(10)(11)(12).Cre has proven to be a valuable tool for manipulating the genomes of mammalian cells and of mice in situ (13-19). As an example, Cre-mediated recombination has all but eliminated the problem of ''selection cassette effects'' associated with some targeted mutations (15). Although Cre is most commonly used to generate short deletions in transgenic mice (13), it has also proven useful for recombination between sites up to several centimorgans apart on individual c...

Disruption of Foxg1 expression by knock-in of Cre recombinase: Effects on the development of the mouse telencephalon

“…More detailed analysis indicated that proliferating, rather than postmitotic, cells were vulnerable to the effects of Cre recombinase, as cryptic recombination events were only observed in mitotically-active cells (Loonstra et al, 2001). There have been two reports of overt pathology in cre-expressing transgenic mice, where high levels of Cre resulted in male infertility when expressed in postmeiotic spermatids (Schmidt et al, 2000) and hydrocephaly when expressed in neuronal progenitors (Forni et al, 2006). In the latter study, the genotoxic effects required the levels of Cre recombinase to reach a critical, high threshold in the cell nucleus, as had been reported previously in vitro (Baba et al, 2005).…”

Characterization of transgene expression and Cre recombinase activity in a panel of Thy‐1 promoter‐Cre transgenic mice

“…For abbreviations, see Table 1. pathologic condition further, however, it is possible that overexpression of Cre could be deleterious to neurons, as Cre expression has been shown to be toxic to cells in culture and in transgenic mice (Schmidt et al, 2000;Loonstra et al, 2001). Gene ablation by using the Cre-loxP system requires that Cre expression is spatially and temporally restricted to the tissue of interest, especially if one is studying the effects of ablating genes whose expression is widespread.…”

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

“…Obviously, poor cre expression in germ line cells cannot be the (only) reason for low or absent transmission of rearrangements via the germ line. Third, Cre toxicity causes somatic phenotypes in tomato , which may pose a problem in gametes especially when expressed to high levels as shown in mammalian cells (Schmidt et al (2000); Loonstra et al (2001)). Selection against Cre containing gametes can explain low over-all transmission frequencies of deletions through the germ line, but Cre toxicity is not expected to differentiate between larger and smaller deletions.…”