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- 积分
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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
- d1 i$ q2 f# b6 B1 b" XKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1/ r& ]# Y( Y9 d( d* k; e
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,, R/ x' r* O" _
Japan# o8 K, X9 V S$ ?
BioTechniques 41:311-316 (September 2006)
- K* Q5 f' S, Y1 a, a7 c8 Xdoi 10.2144/000112233
A2 i+ `" F9 o6 KTargeted gene disruption is a powerful tool for studying gene function in cells and animals.
, ~6 k$ U/ C0 k+ Y Y" xIn addition, this technology includes a potential to correct disease-causing mutations.& ?% f, C; S1 G+ b9 R+ j$ ?8 e
However, constructing targeting vectors is a laborious step in the gene-targeting strategy,
, n- g' m5 K+ }3 ?* Teven apart from the low efficiency of homologous recombination in mammals. Here, we introduce
$ D% {+ z; ~* P1 c8 ?+ xa quick and simplified method to construct targeting vectors. This method is based
4 C" I! e/ P8 {on the commercially available MultiSite Gateway® technology. The sole critical step is to0 o: g& u0 Q) H. c' k7 b
design primers to PCR amplify genomic fragments for homologous DNA arms, after which
5 v4 i! B& q9 O4 r- i6 Z' d2 Hneither ligation reaction nor extensive restriction mapping is necessary at all. The method1 O! [& H7 a0 v% {9 @. c
therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms6 k: s) H' |# j q q7 \" r
whose genome has been sequenced. Recently, we and others have shown that the human pre-' d0 v- ~( \7 C3 U) M
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified
7 v7 C: _' K" z) ^3 n: wvector construction system and the high-efficiency gene targeting in the Nalm-6 cell line
+ _3 m4 J, a. d- t( f; chas enabled rapid disruption of virtually any locus of the human genome within one month,
4 a/ z* W( V& fand homozygous knockout clones lacking a human gene of interest can be created within 2–3( B2 n0 h v6 B: W6 }! q
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
z# O! m5 r7 o- i2 e3 nhuman—genes.
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发表于 2009-3-20 10:16
