Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids

Abstract

The bacteriophage P1 Cre/loxP 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 Cre/loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.

Figure 1

Postmeiotic chromatin disorganization in Prm1-Cre-hGH male mice. (a) Transgene structure. Red indicates sequences derived from the mouse Prm1 gene; yellow denotes the bacteriophage P1 Cre gene; and blue denotes 3′ flanking sequences from the hGH gene. (b) Sperm morphology. High magnification of wild-type and Prm1-Cre-hGH spermatozoa showed no visible differences. The focal plane was set to show the sperm head shape; differences in tail-shaft appearance are because of focal plane. (c) DAPI-stained chromosome spreads of fertilized eggs from matings between wild-type females and either wild-type (Left) or Prm-Cre-hGH males (Right). (d) Meiotic chromosome spreads from wild-type and Prm1-Cre-hGH testes. Testicular sperm at various stages of condensation appear in meiosis II spread fields for stochastic reasons.

Figure 2

Cre^YI-VD mutant. (a) Somatic cell Cre^YI-VD-hGH expression vectors. Colors as in Fig. 1a; green denotes CMV promoter sequences. (b) In vivo recombination assay. A fragment of arbitrary extragenic mouse genomic DNA (light blue) containing two loxP sites (red) flanking a 1.2-kb insert (green) was transiently cotransfected into human 293 cells with either CMV-Cre-hGH (lanes 4–6) or CMV-Cre^YI-VD-hGH. After 48 h, DNA was extracted and analyzed for recombination by using the indicated PCR primers. Lane M, DNA size markers. Lanes 1 and 2, Cells transfected with 2.0 μg CMV-Cre-hGH or CMV-Cre^YI-VD-hGH, respectively. Lane 3, Cells transfected with 1.0 μg substrate plasmid only. Lanes 4–6, Cells cotransfected with 1.0 μg substrate plasmid and 2.0 μg, 0.2 μg, or 0.02 μg of CMV-Cre-hGH expression plasmid, respectively. The similar yield of recombined product in all three lanes indicates that even the lowest amount of CMV-Cre-hGH plasmid yielded 100% recombination in cotransfected cells. Unrecombined substrate in lanes 4–6 likely stems from substrate plasmid that did not enter cells and therefore was not exposed to Cre. Lanes 7–9, Like lanes 4–6, except that CMV-Cre^YI-VD-hGH was used in place of CMV-Cre-hGH. In all cases, Bluescript plasmid vector DNA was used to supplement each transfection to a total of 3.0 μg DNA. (d) Expression of Prm1-Cre-hGH and Prm1-Cre^YI-VD-hGH mRNAs in transgenic mouse testes. RNase protection analyses were performed on the indicated amounts of total RNA harvested from testes of 12- to 14-wk-old wild-type (wt), Prm1-Cre-hGH, or Prm1-Cre^YI-VD-hGH mice. The Cre probe hybridizes to 193 bases of either Cre or Cre^YI-VD mRNA. The endogenous Prm1 mRNA signal served as a normalization control.