Knockout rats via embryo microinjection of zinc-finger nucleases.
The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Geurts
Aron M
AM
Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, WI 52336, USA.
Cost
Gregory J
GJ
Freyvert
Yevgeniy
Y
Zeitler
Bryan
B
Miller
Jeffrey C
JC
Choi
Vivian M
VM
Jenkins
Shirin S
SS
Wood
Adam
A
Cui
Xiaoxia
X
Meng
Xiangdong
X
Vincent
Anna
A
Lam
Stephen
S
Michalkiewicz
Mieczyslaw
M
Schilling
Rebecca
R
Foeckler
Jamie
J
Kalloway
Shawn
S
Weiler
Hartmut
H
Ménoret
Séverine
S
Anegon
Ignacio
I
Davis
Gregory D
GD
Zhang
Lei
L
Rebar
Edward J
EJ
Gregory
Philip D
PD
Urnov
Fyodor D
FD
Jacob
Howard J
HJ
Buelow
Roland
R
D000818:Animals; D001483:Base Sequence; D004247:DNA; D004622:Embryo, Mammalian; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D005260:Female; D055786:Gene Knockout Techniques; D049452:Green Fluorescent Proteins; D007075:Immunoglobulin M / Q000235:genetics*; D008297:Male; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger; D051381:Rats; D016335:Zinc Fingers / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000235:genetics*
D007075:Immunoglobulin M; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C498787:Rab38 protein, rat; D020691:rab GTP-Binding Proteins