Mammalian genomes contain active recombinase recognition sites
Introduction
Genetic engineering of mammalian cells often requires making permanent changes in the genome of target cells (Verma and Somia, 1997). An appealing strategy for permanence would involve site-specific integration of introduced sequences into safe positions in the genome. Homologous recombination can provide a high degree of specificity of position, but its efficiency is low (Vega, 1991). Recombination that is both highly specific and efficient can be mediated by recombinases isolated from microorganisms.
In some cases, recombinases have been shown to function in higher eukaryotic cells. For example, the Cre recombinase derived from E. coli phage P1 works well in mammalian cells (Sauer and Henderson, 1988, Sauer and Henderson, 1990, Sauer, 1994). Cre can mediate either excision or integration, depending on placement of its target loxP sites. The loxP recombination site is 34 bp long and consists of two 13 bp palindromes separated by an asymmetric 8 bp core sequence (Hoess et al., 1982). On a statistical basis, loxP is not expected to occur in mammalian genomes. To use Cre-mediated integration in mammalian cells, loxP sites have been first placed into the genome by imprecise and/or low-frequency procedures (Sauer, 1994). Recombinase-mediated integration would be more attractive for genetic engineering if sites that could act as targets for the Cre recombinase occurred endogenously in mammalian genomes at safe locations, such as regions outside of known genes.
It has been shown that most bases in the loxP core sequence can deviate from wild-type, as long as the cores match between the two recombining lox sites (Hoess et al., 1986). Minor changes in the loxP palindromes have also been shown to allow for Cre function in several cases (Abremski and Hoess, 1985, Abremski et al., 1988, Albert et al., 1995, Sauer, 1996, Sauer, 1992). Statistically, the presence of active lox sites in mammalian genomes requires that more significant departures from the loxP sequence be adequate to allow efficient Cre function. In the E. coli genome, a poor match to the loxP site called loxB occurs, but functions several orders of magnitude less well than loxP (Hoess et al., 1982, Sternberg et al., 1981). Similarly, in the yeast genome, several sites with a poor (10−5) function were identified (Sauer, 1992). Such sites would have little utility for genetic engineering because of their limited efficiency.
More recently, a single site in the yeast genome with approximately a third of the wild-type loxP function was identified (Sauer, 1996). This site has a remarkable degree of homology to loxP, including identity at 21 of 26 positions in the palindromes. It was not clear if the activity of the yeast site derived from fortuitous matching at important bases. On the hypothesis that a sufficiently small subset of the base pairs in the loxP palindromes may be adequate for function, and using genetic, biochemical, and structural data to identify relevant bases, we searched the human and mouse databases for pseudo-lox sites and tested candidate sites for function. We thus identified the first endogenous functional lox sites from higher eukaryotes.
Section snippets
Excision assay plasmids
Plasmid pLCG1 (Fig. 1A) was created by inserting a 4.3 kb XbaI–BspHI fragment from pCMVSPORT-βgal (Life Technologies, Grand Island, NY) containing the lacZ gene, encoding β-galactosidase, driven by the CMV promoter into the EcoRV site of pLitmus29 (New England Biolabs, Beverly, MA), in the orientation opposite to the existing lacZα. This plasmid was then used as a base for the construction of pWTLox2 (Fig. 1B) used in the human excision assay. In general, annealed synthetic oligonucleotides
Computer search for pseudo-lox sites
To search for candidate pseudo-lox (ψlox) sites, we used the findpatterns algorithm of the Wisconsin Software Package Version 9.0 developed by the Genetics Computer Group (GCG; Madison, WI) to screen all sequences in the rodent and primate GenBank databases (Benson et al., 1998). The search was first performed on Release 102.0, 15 August 1997, and updated searches were performed through June 1998. Two different search strategies were used to find sequences with resemblances to the wild-type loxP
Discussion
This study demonstrates that pseudo-lox sites that elicit efficient Cre-mediated recombination exist in mammalian genomes. A computer search, coupled with our predictions about which bases we expected to be more strongly conserved, proved to be adequate to identify pseudo-lox sites that had substantial recombination frequencies. A surprisingly limited number of positions in the 34 bp loxP site are important for DNA recognition and binding. This lower number is still adequate to confer uniqueness
Acknowledgements
We thank Eddie Baba for expert technical assistance and Frank Buchholz and Francis Stewart for the 294-Cre strain of E. coli. We are grateful to Samuel Karlin and Jan Mrazek for assistance with the computer search. This work was supported by grants from the NIH (DK51834) and the Cystic Fibrosis Foundation to M.P.C. Postdoctoral fellowship support from the Stanford Dean's Postdoctoral Fellowship Fund (B.T.) and the Fundação para a Ciência e Tecnologia, Portugal (M.J.G.) is gratefully
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