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Nucleic Acids Research, 2000, Vol. 28, No. 12 E65-e65
© 2000 Oxford University Press

Modification of human ß-globin locus PAC clones by homologous recombination in Escherichia coli

A. M. Ali Imam*, George P. Patrinos, Mariken de Krom, Stefania Bottardi, Rick J. Janssens, Eleni Katsantoni1, Albert W. K. Wai, David J. Sherratt2 and Frank G. Grosveld

Erasmus University Rotterdam, CBG and MGC-Department of Cell Biology, PO Box 1738, 3000 DR Rotterdam, The Netherlands, 1University of Crete, Faculty of Medicine and Institute of Molecular Biology and Biotechnology, PO Box 1527, Heraklion, Crete 71110, Greece and 2Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

We report here modifications of human ß-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.coli RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.

* To whom correspondence should be addressed. Tel: +31 10 408 7164; Fax: +31 10 408 9468; Email: imam@ch1.fgg.eur.nl


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