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Nucleic Acids Research, 2000, Vol. 28, No. 17 e79
© 2000 Oxford University Press

Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli

Mikhail Nefedov, Robert Williamson and Panayiotis A. Ioannou*

CAGT Research Group, The Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria 3052, Australia

We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact ß-globin locus in a second generation BAC vector. We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the ß-globin gene. All recombinant clones examined contained the TetR gene at the correct target site. Next, a PCR fragment with the IVS I-110 G->A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination. Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid. Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened. Analysis of the recombinant clones did not show any other modifications or rearrangements. Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.

* To whom correspondence should be addressed. Tel: +61 3 8341 6232; Fax: +61 3 9348 1391; Email: ioannoup@cryptic.rch.unimelb.edu.au


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