Development of a novel bacterial artificial chromosome cloning system for functional studies.
Bacterial artificial chromosome (BAC) cloning systems currently in use generate high quality genomic libraries for gene mapping, identification, and sequencing. However, the most commonly used BAC cloning systems do not facilitate functional studies in eukaryotic cells. To overcome this limitation, we have developed pEBAC190G, a new BAC vector that combines the features of the first generation PAC/BAC vectors with eukaryotic elements that facilitate the transfection, episomal maintenance, and functional analysis of large genomic fragments in eukaryotic cells. A number of different cloning strategies may be used to retrofit genomic fragments from existing libraries into the new vector. The system was tested by the retrofitting of a 170kb NotI genomic fragment from the RPCI-11 BAC library into the NotI site of pEBAC190G. Clones from any eukaryotic genomic library harboured in this vector can be transferred from bacteria directly to eukaryotic cells for functional analysis.
CAGT Research Group, The Murdoch Childrens Research Institute, University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia.
Keith
K
Al-Hasani
Kim
K
Simpfendorfer
Hady
H
Wardan
Jim
J
Vadolas
Faten
F
Zaibak
Ricardo
R
Villain
Panayiotis A
PA
Ioannou
D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005057:Eukaryotic Cells; D015723:Gene Library; D005822:Genetic Vectors; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic
D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D008765:Methylmalonyl-CoA Mutase