High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.
One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.
Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill Road, Tarrytown, New York 10591, USA.
David M
DM
Valenzuela
Andrew J
AJ
Murphy
David
D
Frendewey
Nicholas W
NW
Gale
Aris N
AN
Economides
Wojtek
W
Auerbach
William T
WT
Poueymirou
Niels C
NC
Adams
Jose
J
Rojas
Jason
J
Yasenchak
Rostislav
R
Chernomorsky
Marylene
M
Boucher
Andrea L
AL
Elsasser
Lakeisha
L
Esau
Jenny
J
Zheng
Jennifer A
JA
Griffiths
Xiaorong
X
Wang
Hong
H
Su
Yingzi
Y
Xue
Melissa G
MG
Dominguez
Irene
I
Noguera
Richard
R
Torres
Lynn E
LE
Macdonald
A Francis
AF
Stewart
Thomas M
TM
DeChiara
George D
GD
Yancopoulos
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D051379:Mice / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods; D016297:Mutagenesis, Site-Directed; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism
D011994:Recombinant Proteins