Group leader: 

Andrew Smith

Contact: 

Andrew [dot] Smith [at] ed [dot] ac [dot] uk

Background
The focus of my groups work carried out at the MRC Laboratory of Molecular Biology prior to joining the Centre has been to demonstrate the feasibility of constructing large scale chromosomal rearrangements in the multi-megabase size range using a combination of gene targeting and site-specific recombination (Smith, A.J.H. et al., (1995 NatureGenetics 9, 376-385). In this strategy homologous recombination is used to deliver to the desired locations in the genome short DNA sequences, loxP sites, which are sites at which a recombinase, Cre,cleaves and rejoins DNA to generate recombinant molecules. Recombination between the loxP sites occurs when the Cre recombinase is transiently expressed and selection for this event can be achieved in HPRT+ cells by arranging for the Cre-mediated recombination event to reconstruct a functional human Hprt minigene from two defective segments flanking the loxP sites.

We generated a chromosomal translocation by targeting the loxP sites to two different chromosomes (12 and 15) and then recovering HPRT+ cells after transient Cre expression. HPRT+ clones were obtained at a frequency of10-7 to 10-8and were shown to contain the predicted translocation event. Thus this strategy should be generally applicable for engineering a variety of different types of chromosomal rearrangement in mice which would represent a new technical achievement for mammalian transgenesis with significant applications. The Bradley group at Baylor in Houston, USA have now used a similar strategy to transmit megabase size deletions into the germ line (Ramirez-Solis, R. et al., (1995) Nature 378, 720-724).

Aims
Our aims at the Centre are to exploit this technology to facilitate new approaches for genetic analysis of the mouse genome, to generate model transgenic systems which will recapitulate chromosomal translocations identified in human leukaemias, and to construct recombinant stem cell lines with grafted syntenic chromosomal regions from other species or other mouse strains.

FISH analysis using a chromosome 9 specific paint (pink) and a 3pk-Hyal1 deletion interval specific paint (yellow) of targeted ES cells before (A) and after (B) Cre recombination to show loss of the 3p21.2 region.

Selected publications

• Wallace HAC, Marques-Kranc F, Richardson M, Luna-Crespo F, Sharpe JA, Hughes J, Wood WG, Higgs DR, Smith AJH. 2007. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell 128:197-209.
• Testa et al. 2003. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21:443-447.
• Smith et al. 2002. Cre-loxP chromosome engineering of a targeted deletion in the mouse corresponding to the 3p21.3 region of homozygous loss in human tumours. Oncogene 21:4521-4529.
• Smith et al. 1995. A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination. Nature Genetics 9:378-384.