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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,& _# c/ U! N, }3 Z3 Y
Kei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama14 |0 V: w5 L: n6 m
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,
/ C9 f+ F2 z4 [+ z% x& zJapan
3 [. j! u- v: G) X2 ]2 W' d% ~5 X( {; HBioTechniques 41:311-316 (September 2006)$ J' Q& c, h2 @! b: j. j
doi 10.2144/000112233
- x% Z" `+ A& L. K. GTargeted gene disruption is a powerful tool for studying gene function in cells and animals.
' d7 K2 r' J9 i$ h! CIn addition, this technology includes a potential to correct disease-causing mutations.
1 q6 x# b9 H, B8 }However, constructing targeting vectors is a laborious step in the gene-targeting strategy,6 D4 E! q3 p# H. F% t6 a' \
even apart from the low efficiency of homologous recombination in mammals. Here, we introduce0 [. t9 v# `6 Y9 P# p' t* D3 S
a quick and simplified method to construct targeting vectors. This method is based) [, J( C9 W& O6 H+ r2 [
on the commercially available MultiSite Gateway® technology. The sole critical step is to
& U" ^1 ~; p6 i6 J6 K0 Y1 ]design primers to PCR amplify genomic fragments for homologous DNA arms, after which% T/ j7 x: Q4 j! D {% t/ B' p6 `
neither ligation reaction nor extensive restriction mapping is necessary at all. The method9 S7 `% q/ e; X& c! g
therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms! {4 ^. z, v& o7 U8 n
whose genome has been sequenced. Recently, we and others have shown that the human pre-* p& {9 X/ [8 }8 K, n0 u
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified, Z' ]9 l* A+ C3 V6 o) o u
vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line& ~3 w+ l1 ~; E# C7 Q% K( u% w# S
has enabled rapid disruption of virtually any locus of the human genome within one month,. m5 }7 G- I3 l. ?; s6 X+ g
and homozygous knockout clones lacking a human gene of interest can be created within 2–3: e/ x" \* P# m4 f2 H2 e4 s
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
* N2 S* [: t) H& @0 v7 S7 n9 |human—genes.+ ]% f% ]! ? ]' n0 F% @; d
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
