Sp R I I - Ppo I I - Ppo I I - Ppo Subcloning of the two BAC inserts Linearisation with I - I Ppo BAC 1

Background: BAC clones containing entire mammalian genes including all the transcribed region and long range controlling elements are very useful for functional analysis. Sequenced BACs are available for most of the human and mouse genomes and in many cases these contain intact genes. However, large genes often span more than one BAC, and single BACs covering the entire region of interest are not available. Here we describe a system for linking two or more overlapping BACs into a single clone by homologous recombination. Results: The method was used to link a 61-kb insert carrying the final 5 exons of the human CFTR gene onto a 160-kb BAC carrying the first 22 exons. Two rounds of homologous recombination were carried out in the EL350 strain of bacteria which can be induced for the Red genes. In the first round, the inserts of the two overlapping BACs were subcloned into modified BAC vectors using homologous recombination. In the second round, the BAC to be added was linearised with the very rare-cutting enzyme I-PpoI and electroporated into recombination efficient EL350 bacteria carrying the other BAC. Recombined BACs were identified by antibiotic selection and PCR screening and 10% of clones contained the correctly recombined 220-kb BAC. Conclusion: The system can be used to link the inserts from any overlapping BAC or PAC clones. The original orientation of the inserts is not important and desired regions of the inserts can be selected. The size limit for the fragments recombined may be larger than the 61 kb used here and multiple BACs in a contig could be combined by alternating use of the two pBACLink vectors. This system should be of use to many investigators wishing to carry out functional analysis on large mammalian genes which are not available in single BAC clones. Background Sequenced BAC and PAC clones are now available for most of the human and mouse genomes and have proven extremely useful in the analysis of gene function. The large insert size of the clones allow many mammalian genes to be cloned intact with all the long range controlling elements and BAC transgenics have generally given physiological levels of tissue specific expression in transgenic mice (for example [1,2]). In addition to the sequenced BACs, there are also databases of BAC end sequences http://www.tigr.org/tdb/humgen/bac_end_search/ bac_end_intro.html which allow one to locate additional BACs covering a specific region. However, there are many large mammalian genes which are of the same order of size, or larger, than the average insert size of the BAC libraries (about 170-kb) and for these it is often difficult to find a single BAC spanning the entire gene and controlling elements. For these genes, large gene clusters and Published: 06 January 2004 BMC Biotechnology 2004, 4:1 Received: 28 October 2003 Accepted: 06 January 2004 This article is available from: http://www.biomedcentral.com/1472-6750/4/1 © 2004 Kotzamanis and Huxley; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.