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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,& q1 Z( w) }% \+ J0 x9 W
Kei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1# O, m# x: F* J7 a) ^, U& @ L
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,
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BioTechniques 41:311-316 (September 2006)' p! y4 |! |) X7 S- v8 a
doi 10.2144/000112233! d4 z- U: W o& u4 [. A
Targeted gene disruption is a powerful tool for studying gene function in cells and animals.
: p7 m% ]/ A3 h0 V/ g+ tIn addition, this technology includes a potential to correct disease-causing mutations.
* y. x, f" Q) G- w0 ~& V" ?However, constructing targeting vectors is a laborious step in the gene-targeting strategy,
* K: ?5 h8 h2 E, Xeven apart from the low efficiency of homologous recombination in mammals. Here, we introduce4 r4 n+ h, U! j! i
a quick and simplified method to construct targeting vectors. This method is based1 H9 h3 o- F9 H( _4 B: c' e! z
on the commercially available MultiSite Gateway® technology. The sole critical step is to
' m1 }+ C, h. udesign primers to PCR amplify genomic fragments for homologous DNA arms, after which
3 I$ W) W. ^% W4 `; n7 c* Xneither ligation reaction nor extensive restriction mapping is necessary at all. The method
, U$ F+ T9 p; {5 E2 P" Itherefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms0 W' a y b, b5 A: C- i
whose genome has been sequenced. Recently, we and others have shown that the human pre-
$ p5 s/ \. Q2 F+ O% P& N; YB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified
$ p9 `; w! B& i$ |* jvector construction system and the high-efficiency gene targeting in the Nalm-6 cell line" S( o8 d: p" m* p: d5 O+ ?
has enabled rapid disruption of virtually any locus of the human genome within one month,5 o7 w! g ?$ B6 `9 ~- q0 S
and homozygous knockout clones lacking a human gene of interest can be created within 2–3) W. c0 P7 I# c- r' E: k" }/ ?
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
, J; o t# p' d0 N0 |- \, U* ^human—genes.
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
