Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis.
The availability of genomic sequence information and extensive bacterial artificial chromosome (BAC) libraries for both the mouse and human genomes is ushering in a new era in biological research and disease modeling. To facilitate the study of large mammalian genes in vivo, we have developed: i) a simple lambda bacteriophage-based methodology for recombining overlapping bacterial artificial chromosomes (BACs) into a single larger BAC, and ii) a new methodology for targeting "seamless" mutations into BACs. In the first method, overlapping sequence from one BAC is cloned alongside a selectable marker and placed between unique sequence arms from the terminus of the other BAC to create a targeting construct. Two rounds of recombination-based cloning are then performed. The robustness of this methodology is demonstrated herein by using it to obtain a 254 kb BAC containing the entire human androgen receptor (hAR) gene. In the second method, transient expression of three lambda bacteriophage genes to 'pop-in' a targeting cassette is followed by RecA expression from the targeting vector itself to 'pop-out' the vector backbone. This new "hybrid recombineering" method combines the strengths of the lambda bacteriophage and RecA systems, while avoiding their major weaknesses. Application of this method for introduction of a 162 CAG repeat expansion into the hAR 254kb BAC is shown. With "hybrid recombineering", we believe that the power and utility of the classical 'pop-in/pop-out' approach -- so commonly and efficiently employed in yeast for decades -- can now be achieved with BACs.
Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA 98195, USA.
Bryce L
BL
Sopher
Albert R
AR
La Spada
D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics; D011944:Receptors, Androgen / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*
D011944:Receptors, Androgen; D011938:Rec A Recombinases