Manipulating large genomic clones via in vivo recombination in bacteria.
Transgenesis is proving to be a powerful technique in studying the molecular genetics of hypertension. The ability to target specific mutations resulting in either loss of function, by gene deletion, the insertion of reporter sequences, or the subtle change of function by nucleotide replacement, can facilitate the understanding of gene function and its role in the manifestation of diseases. However an inherent problem associated with transgenic studies is the lack of consistent expression observed between independent lines of animals which have integrated the same transgene, a phenomenon known as 'position effect'. Small transgenes are almost invariably subject to position effect due to the absence of essential regulatory elements required to maintain an open chromatin structure. This phenomenon may be overcome if larger transgenes, isolated using vectors such as yeast artifical chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-based vectors, are used. Studies using such transgenes have reported levels of expression which are consistent between lines and dependent upon the number of copies integrated. The introduction of modifications into these large genomic clones is not practical by traditional restriction endonuclease strategies and so is dependent upon in vivo recombination to maintain structural integrity. Here we demonstrate the modification of a 100 Kb P1 clone spanning the renin locus using the BAC targeting strategy described by Yang et al (Nat Biotechnol 1997; 15: 859-865).
Payne
C M
CM
Centre for Genome Research, The Kings Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.
Mullins
L J
LJ
Mullins
J J
JJ
D000818:Animals; D017101:Bacteriophage P1 / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D011995:Recombination, Genetic / Q000235:genetics / Q000502:physiology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity