A randomized library approach to identifying functional lox site domains for the Cre recombinase.
The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.
Sheren
Jamie
J
University of Colorado, Department of Molecular, Cellular and Developmental Biology, 347 UCB, Boulder, CO 80309-0347, USA. sheren@colorado.edu
Langer
Stephen J
SJ
Leinwand
Leslie A
LA
D001287:Attachment Sites, Microbiological; D015723:Gene Library; D019426:Integrases / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
C045073:Cre recombinase; D019426:Integrases
Gene. 2000 Feb 22;244(1-2):47-54
10689186
Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7905-9
1518811
Nucleic Acids Res. 2002 Jul 1;30(13):2764-71
12087159
Nucleic Acids Res. 2002 Jul 15;30(14):3067-77
12136089
Anal Biochem. 2002 Sep 1;308(1):90-9
12234468
J Mol Biol. 1981 Aug 25;150(4):467-86
6276557
J Mol Biol. 1981 Aug 25;150(4):487-507
6276558
Proc Natl Acad Sci U S A. 1982 Jun;79(11):3398-402
6954485
Cell. 1983 Apr;32(4):1301-11
6220808
J Biol Chem. 1984 Feb 10;259(3):1509-14
6319400
Proc Natl Acad Sci U S A. 1984 Feb;81(4):1026-9
6230671
Nucleic Acids Res. 1986 Mar 11;14(5):2287-300
3457367
J Mol Biol. 1990 Dec 20;216(4):873-82
2266559
New Biol. 1990 May;2(5):441-9
2288914
J Mol Biol. 1992 Feb 20;223(4):911-28
1554399
Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6232-6
1631115
Nucleic Acids Res. 1992 Sep 11;20(17):4451-5
1408747
Nucleic Acids Res. 1993 May 11;21(9):2265-6
8502579
Cell. 1993 Jun 18;73(6):1155-64
8513499
Trends Genet. 1993 Dec;9(12):413-21
8122308
Science. 1994 Jul 1;265(5168):103-6
8016642
Plant J. 1995 Apr;7(4):649-59
7742860
Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6991-5
7624356
Science. 1995 Sep 8;269(5229):1427-9
7660125
Gene. 1996 Jun 12;172(1):59-63
8654992
Nucleic Acids Res. 1996 Aug 1;24(15):3118-9
8760904
Nucleic Acids Res. 1996 Dec 1;24(23):4608-13
8972844
Gene. 1996 Dec 12;183(1-2):53-60
8996087
Nature. 1997 Sep 4;389(6646):40-6
9288963
Methods. 1998 Apr;14(4):381-92
9608509
Gene. 1998 Aug 17;216(1):55-65
9714735
J Biol Chem. 1998 Sep 4;273(36):22884-91
9722507
J Mol Biol. 1998 Nov 27;284(2):363-84
9813124
BMC Genomics. 2006;7:73
16595017
J Mol Biol. 1985 Feb 5;181(3):351-62
3856690
Gene. 2000 Jun 13;251(1):91-9
10863100