Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis Export

@article{citeulike:570710, abstract = {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 254 kb 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.}, author = {Sopher, Bryce L. and La Spada, Albert R.}, citeulike-article-id = {570710}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.gene.2005.11.034}, citeulike-linkout-1 = {http://www.sciencedirect.com/science/article/B6T39-4J91R5C-1/2/dd1092aaf323dc39e24bd940c2d3f3fc}, day = {12}, doi = {10.1016/j.gene.2005.11.034}, journal = {Gene}, keywords = {allelic-recombination, bac}, month = {April}, number = {1}, pages = {136--143}, posted-at = {2006-03-30 13:37:18}, priority = {2}, title = {Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis}, url = {http://dx.doi.org/10.1016/j.gene.2005.11.034}, volume = {371}, year = {2006} }

TY - JOUR ID - citeulike:570710 L3 - citeulike-article-id:570710 N2 - 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 254 kb 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. IS - 1 JF - Gene EP - 143 TI - Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis VL - 371 SP - 136 KW - allelic-recombination KW - bac AU - Sopher, Bryce AU - La Spada, Albert PY - 2006/04/12/ UR - http://dx.doi.org/10.1016/j.gene.2005.11.034 ER -