Mini-lambda: a tractable system for chromosome and BAC engineering.
The bacteriophage lambda (lambda) recombination system Red has been used for engineering large DNA fragments cloned into P1 and bacterial artificial chromosomes (BAC or PAC) vectors. So far, this recombination system has been utilized by transferring the BAC or PAC clones into bacterial cells that harbor a defective lambda prophage. Here we describe the generation of a mini-lambda DNA that can provide the Red recombination functions and can be easily introduced by electroporation into any E. coli strain, including the DH10B-carrying BACs or PACs. The mini-lambda DNA integrates into the bacterial chromosome as a defective prophage. In addition, since it retains attachment sites, it can be excised out to cure the cells of the phage DNA. We describe here the use of the mini-lambda recombination system for BAC modification by introducing a selectable marker into the vector sequence of a BAC clone. In addition, using the mini-lambda, we create a single missense mutation in the human BRCA2 gene cloned in a BAC without the use of any selectable marker. The ability to generate recombinants very efficiently demonstrates the usefulness of the mini-lambda as a very simple mobile system for in vivo genome engineering by homologous recombination, a process named recombineering.
Court
Donald L
DL
Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, NCI/FCRDC, National Cancer Institute at Frederick, Building 539/Room 243, P.O. Box B, Frederick, MD 21702, USA. court@ncifcrf.gov
Swaminathan
Srividya
S
Yu
Daiguan
D
Wilson
Helen
H
Baker
Teresa
T
Bubunenko
Mikail
M
Sawitzke
James
J
Sharan
Shyam K
SK
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005822:Genetic Vectors / Q000235:genetics; D017354:Point Mutation; D039002:Prophages / Q000235:genetics; D011995:Recombination, Genetic
D004269:DNA, Bacterial