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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
3 n* y' |9 `: x8 oKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1
) R3 P2 A* Q$ t9 f# H( ` N1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,1 H6 B* X" U$ `
Japan; n% j+ P- F) j: S& r% u! Z
BioTechniques 41:311-316 (September 2006)
4 J! D; L6 |, {# s/ d' W( vdoi 10.2144/000112233
, y. \& I1 X& n8 Z) k7 U( S ZTargeted gene disruption is a powerful tool for studying gene function in cells and animals.2 | _/ Y& s" D/ }
In addition, this technology includes a potential to correct disease-causing mutations.
( x' v/ @: `/ YHowever, constructing targeting vectors is a laborious step in the gene-targeting strategy,2 {# N1 Z/ Z, ?: M
even apart from the low efficiency of homologous recombination in mammals. Here, we introduce4 T* g. ?, @" g
a quick and simplified method to construct targeting vectors. This method is based0 ?" t# W# P# V: |6 l
on the commercially available MultiSite Gateway® technology. The sole critical step is to" S- X) e' s( i! T2 v7 ^' u
design primers to PCR amplify genomic fragments for homologous DNA arms, after which
l( l# Q# S) o' l) f2 `neither ligation reaction nor extensive restriction mapping is necessary at all. The method
% X! z4 v4 y: a& R2 _1 Ntherefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms9 N- E8 Z4 g& E0 N* M# S8 `( b
whose genome has been sequenced. Recently, we and others have shown that the human pre-6 }, o+ l; {7 s) U
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified1 k$ z. X5 ]3 K8 ]$ |
vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line( j* _* u! a, }0 ]
has enabled rapid disruption of virtually any locus of the human genome within one month,
2 _. Z @) ?; ~* ^- |/ \& s- Iand homozygous knockout clones lacking a human gene of interest can be created within 2–3
. C& j0 G9 N6 pmonths. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
- w* D3 h5 V" r" `/ Lhuman—genes.
0 ~$ W$ W/ c6 w" g8 r3 ?' }$ k% U! j! E }& J- i/ J. L
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
