I-SIS has predicted that the recombinase enzyme used in terminator technologies will scrambled genomes (see Terminator in new guises I-SIS News #3, December 1999). This has now been demonstrated in transgenic mice engineered with the recombinase.
The recombinase Cre is part of the 'site-specific recombination' Cre/lox system originally isolated from the bacteriophage (bacterial virus) P1. Cre catalyses recombination between two lox sites, splicing out any stretch of DNA in between. The lox site is a 34 basepair element consisting of 13 basepair inverted repeat separated by a core of 8 basepairs. In order to work, the 8 basepair core of the two lox sites have to be in the same orientation.
The system is not only used in plants, but extensively exploited in transgenic mice. Studies in the test-tube have shown that Cre recombinase can catalyze recom-bination between DNA sequences found naturally in yeast and mammalian genomes. These 'illegitimate sites' often bear little sequence similarity to the lox element. However, there have been no reports on such illegitimate recognition in the animals or plants themselves. And there have even been pilot studies using the Cre/lox system in human gene therapy.
In a new study [1] researchers in the United States showed that high levels of Cre expression in the sperm cells of heterozygous transgenic mice leads to 100% sterility in the males, despite the absence of any lox sites. Heterozygous mice carry only one copy of the Cre recombinase gene.
The sterility is caused directly by the recombinase enzyme scrambling the genome, essentially by breaking and rejoining DNA at inappropriate sites on the same or different chromosomes. The researchers have pinpointed the genome scrambling event to the time at which the two 'daughter' spermatids (precursors of sperms) and their paired chromosomes have just separated from each other; but are still joined by a 'cytoplasmic bridge'. This is enough to allow the enzyme to pass from the spermatid containing the recombinase gene to the other which does not, thereby to scramble up the chromosomes of both the transgenic and nontransgenic spermatid. The result is 100% sterility. Embryos fertilized by these sperms arrest predominantly at the 2 cell stage, and do not go beyond the four cell stage.
The researchers warn: "These results indicate that Cre can catalyze illegitimate recombination having overt pathological consequences in animals." A similar recombination system is found in animals containing the RAG recombinases. There, illegitimate recombinations in somatic cells are linked to human leukemias.
1.Schmidt, E.E., Taylor, D.S., Prigge, J.R., Barnett, S. and Capecchi, M.R. (2000). Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. PNAS 97, 13702-13707.