Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus.
Transgenesis using large DNA such as YAC or BAC has extended the range of applications in functional genomics. Here we describe an efficient BAC transgenesis protocol using a simple BAC DNA preparation method adopted from YAC DNA purification methods. This method allowed us to isolate BAC DNA from small scale culture of BAC-containing cells in sufficient quantity and purity for microinjection. More than 40 founders have been produced with linearized BAC DNA prepared by this method, and 85% of them contained intact BAC transgenes. In contrast, when circular BAC DNA was injected, an approximately three-fold reduction of transgene integration rate was observed and fewer intact transgene integrations were obtained. A line of transgenic mice carrying a 170-kb BAC clone generated in this way successfully rescued tail and embryonic lethality phenotypes of the mouse Brachyury (T) mutants, further demonstrating the utility of this method in functional analysis of the mouse genome.
Abe
Kuniya
K
Research & Development Team for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan.
Hazama
Masaaki
M
Katoh
Hideki
H
Yamamura
Ken-ichi
K
Suzuki
Misao
M
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D004247:DNA; D005326:Fetal Proteins / Q000235:genetics; D018014:Gene Transfer Techniques; D051379:Mice; D020825:T-Box Domain Proteins / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D000098908:Brachyury Protein
D004247:DNA; D005326:Fetal Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein