RT Journal
A1 Cotta-de-Almeida, Vinícius
A1 Schonhoff, Susan
A1 Shibata, Tomoyuki
A1 Leiter, Andrew
A1 Snapper, Scott B.
T1 A New Method for Rapidly Generating Gene-Targeting Vectors by Engineering BACs Through Homologous Recombination in Bacteria
JF Genome Research 
JO Genome Research 
YR 2003 
FD September 01 
VO 13 
IS 9 
SP 2190 
OP 2194 
DO 10.1101/gr.1356503 
UL http://genome.cshlp.org/content/13/9/2190.abstract 
AB Generating knockout mice is still an expensive and highly time-consuming  process. Target construct generation, the first labor-intensive step in this  process, requires the manipulation of large fragments of DNA and numerous, and  often cumbersome, cloning steps. Here we show the development of a rapid  approach for generating targeting constructs that capitalizes on efficient  homologous recombination between linear DNA fragments and circular plasmids in  Escherichia coli (“recombineering”), the availability of  bacterial artificial chromosomes (BACs), and the accessibility of the sequence  of the mouse genome. Employing recombineering, we demonstrate with only  1–2 template plasmids, short homologies (40–50bp) between donor  and target DNA, and one subcloning step that we can efficiently manipulate  BACs in situ to generate a complicated targeting vector. This procedure avoids  the need to construct or screen genomic libraries and permits the generation  of most standard, conditional, or knock-in targeting vectors, often within two  weeks.