Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse.
An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.
Wilson
C J
CJ
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Guglielmo
C
C
Moua
N D
ND
Tudor
M
M
Grosveld
G
G
Young
R A
RA
Murray
P J
PJ
D000818:Animals; D018244:Chromosomes, Artificial, Yeast; D004926:Escherichia coli; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL / Q000235:genetics*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013234:Stem Cells / Q000502:physiology; D014162:Transfection