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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
' a4 r5 F- }! b9 F& zKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1
% }2 B0 H: x* O" g! o! y1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,
5 q1 L# |- P% T4 VJapan* J5 L2 [- P! z$ d
BioTechniques 41:311-316 (September 2006)
1 j' P+ ]7 {; Z0 N$ j' `doi 10.2144/000112233
' D( r& D, J! v0 u5 {' WTargeted gene disruption is a powerful tool for studying gene function in cells and animals.
, e [# j7 y9 HIn addition, this technology includes a potential to correct disease-causing mutations.
1 C$ c T" T8 Z, K' Q6 f2 q# IHowever, constructing targeting vectors is a laborious step in the gene-targeting strategy,
9 f2 x5 ]# C) r) y+ R1 p/ seven apart from the low efficiency of homologous recombination in mammals. Here, we introduce
% K* t9 P! {6 K: J' t4 [ A: @a quick and simplified method to construct targeting vectors. This method is based
- j2 z3 E" K n7 v5 o4 bon the commercially available MultiSite Gateway® technology. The sole critical step is to4 W3 j( M3 [, X5 m% S) w# ]% Y
design primers to PCR amplify genomic fragments for homologous DNA arms, after which% M# c5 g5 Q; h
neither ligation reaction nor extensive restriction mapping is necessary at all. The method" F T6 B6 b; A4 k* k
therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms
& n4 x5 e8 K% S- _% o; i9 `- H- ?whose genome has been sequenced. Recently, we and others have shown that the human pre- G* V5 T" h- V( F& q
B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified) a6 v% O7 B6 Z" ~
vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line9 V9 a8 z2 T; Z+ V
has enabled rapid disruption of virtually any locus of the human genome within one month," g$ d$ {/ W0 R0 c; s; M8 M
and homozygous knockout clones lacking a human gene of interest can be created within 2–3/ f% U6 `: r! @
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
2 ?+ [0 c% I! O- a) N3 Ghuman—genes.
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
