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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
$ [) g+ y0 X$ J) m9 I9 IKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1" e8 H$ B+ k6 _% V" F; @9 z
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,: Q+ ^' C2 K" A+ g# U6 G( |! I
Japan4 }# z8 @8 k! s; L5 D
BioTechniques 41:311-316 (September 2006)
" a' R0 A! _. \. E6 i: _doi 10.2144/000112233
/ b% g$ e b4 fTargeted gene disruption is a powerful tool for studying gene function in cells and animals., G; \7 O2 I8 V$ q8 V
In addition, this technology includes a potential to correct disease-causing mutations.
( D9 z6 ]7 U7 W) T9 B) D, sHowever, constructing targeting vectors is a laborious step in the gene-targeting strategy,
! e6 O/ D9 T. Reven apart from the low efficiency of homologous recombination in mammals. Here, we introduce0 ?9 E% x7 K) Q5 \6 f" z7 U% S, S
a quick and simplified method to construct targeting vectors. This method is based1 V- K# n& P3 ]0 q8 I9 x1 o
on the commercially available MultiSite Gateway® technology. The sole critical step is to4 ~& M* g9 D7 @" n! \2 f9 d
design primers to PCR amplify genomic fragments for homologous DNA arms, after which
6 }! G ]3 K& U- S0 U* V/ ineither ligation reaction nor extensive restriction mapping is necessary at all. The method
8 o$ A5 k/ ^ H7 A& _6 N. \4 y ?0 Ntherefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms
& W B" r! e# o3 S5 |, i0 V' J! `whose genome has been sequenced. Recently, we and others have shown that the human pre-3 l: N2 Q) S2 {6 k0 N9 l3 E
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified
$ o+ q+ Q5 r( T+ ^) [# x8 bvector construction system and the high-efficiency gene targeting in the Nalm-6 cell line
" y8 j Z h9 @) Bhas enabled rapid disruption of virtually any locus of the human genome within one month,% x9 Y& ?) v( u! N! _
and homozygous knockout clones lacking a human gene of interest can be created within 2–3
& l% l- Y J. c; [months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
1 ^# m% U& w- v- rhuman—genes.
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
