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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
8 d! q) v5 @ ?, [" LKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1
2 K" V2 H4 J/ \- q/ q. R' z/ I$ U1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,
% X5 U: y4 f9 V! R, f) YJapan: \ v' L8 Q2 K4 {7 ]
BioTechniques 41:311-316 (September 2006)
7 w& E+ O! R+ T0 ydoi 10.2144/000112233 N8 i/ U! J; L! H
Targeted gene disruption is a powerful tool for studying gene function in cells and animals.
2 K" i+ Z) Y0 v7 E6 q/ cIn addition, this technology includes a potential to correct disease-causing mutations.* p' b- [3 o: F7 V. E
However, constructing targeting vectors is a laborious step in the gene-targeting strategy,3 y4 E1 L, m3 V! b0 z4 j/ H
even apart from the low efficiency of homologous recombination in mammals. Here, we introduce
9 p3 B( B |& j. {. V! @a quick and simplified method to construct targeting vectors. This method is based- Q! l& g- L, R" f
on the commercially available MultiSite Gateway® technology. The sole critical step is to6 A( N0 l1 J' o5 p$ @
design primers to PCR amplify genomic fragments for homologous DNA arms, after which
: f3 {: M- n8 s% Eneither ligation reaction nor extensive restriction mapping is necessary at all. The method. S9 G1 q* @: b6 J% h
therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms! k8 q2 `6 j" y0 Y' p0 f+ u* h J! D
whose genome has been sequenced. Recently, we and others have shown that the human pre-
. S+ t8 X3 d* h3 ~. cB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified7 Q* ?( a, o6 l
vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line/ t! H6 v& f' o% M K7 C5 A
has enabled rapid disruption of virtually any locus of the human genome within one month,8 m9 R3 j J! x& ^( N0 N1 t
and homozygous knockout clones lacking a human gene of interest can be created within 2–3
: X0 E4 J; {0 \& v: Qmonths. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly3 K% G- R8 W. {, K% _
human—genes.
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发表于 2009-3-20 10:16
