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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
7 n: ?5 i& X$ A& VKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1+ p: A/ v, {* X! ]0 y: b
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,
6 N) U5 y! V) ?Japan
( p1 b# Y9 k; X- O8 M6 H0 cBioTechniques 41:311-316 (September 2006)7 E0 C$ w# S5 ]6 H
doi 10.2144/000112233: q: n3 g5 @: M% G E
Targeted gene disruption is a powerful tool for studying gene function in cells and animals.
9 @! v! C2 P: ]8 ]In addition, this technology includes a potential to correct disease-causing mutations.) S, u4 P" n! E8 ~( e0 F7 f5 a5 H
However, constructing targeting vectors is a laborious step in the gene-targeting strategy,4 A- z' m* P$ X. p( h
even apart from the low efficiency of homologous recombination in mammals. Here, we introduce0 X0 L, _% X! @; k* p* O
a quick and simplified method to construct targeting vectors. This method is based# N' L3 V7 B( e+ j( W8 \7 Z
on the commercially available MultiSite Gateway® technology. The sole critical step is to
- @' i; q' A* X2 r2 \& @# A Pdesign primers to PCR amplify genomic fragments for homologous DNA arms, after which
* T5 [9 k- z$ J4 a) jneither ligation reaction nor extensive restriction mapping is necessary at all. The method
0 ^' X1 r* {& N) S$ Qtherefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms
1 p% t1 [7 E. @3 q# dwhose genome has been sequenced. Recently, we and others have shown that the human pre-+ y2 y; L( H0 _2 O
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified
2 x+ M ~& b" f" Q& E# I# @8 o: Gvector construction system and the high-efficiency gene targeting in the Nalm-6 cell line
+ |2 e* W2 T% }$ a v+ N- Thas enabled rapid disruption of virtually any locus of the human genome within one month,# {1 v+ R2 R* g( ~% e
and homozygous knockout clones lacking a human gene of interest can be created within 2–3
/ A; p# w6 j1 W- omonths. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
/ E& L6 h+ v* r! Ohuman—genes.
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
