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Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
* @7 j( s- D% L/ ~+ S( i$ kKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1 I; e3 W! l8 a& c: w/ F
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,% W7 V& L. B: F( x. ~8 g
Japan) t# |$ p2 L0 _1 [! f( a
BioTechniques 41:311-316 (September 2006)+ ~# O& H1 U6 y- c( E
doi 10.2144/000112233
) }0 ^6 `5 @9 S; wTargeted gene disruption is a powerful tool for studying gene function in cells and animals.
9 T4 M9 e. ~: Q# H$ H* G( rIn addition, this technology includes a potential to correct disease-causing mutations.
0 I* C2 |) B8 d& e, C) `+ d& HHowever, constructing targeting vectors is a laborious step in the gene-targeting strategy,
8 H. [- }; q- }" ^even apart from the low efficiency of homologous recombination in mammals. Here, we introduce6 K, p/ r8 a" M/ a6 s
a quick and simplified method to construct targeting vectors. This method is based
" E, U, U7 q1 ~* ?on the commercially available MultiSite Gateway® technology. The sole critical step is to3 R. b6 l7 q$ V7 w8 n% ?/ U; [4 \
design primers to PCR amplify genomic fragments for homologous DNA arms, after which
^; M# p! F( ~& x# W6 I+ fneither ligation reaction nor extensive restriction mapping is necessary at all. The method
. X. q# V6 q m6 a+ j- Qtherefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms7 z' F$ B) j' y( |* B R* y' v
whose genome has been sequenced. Recently, we and others have shown that the human pre-
+ ~* E' C. O* {, OB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified5 g e. j! V0 o" `# ]( R- F
vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line
1 {! q" k; A1 m( x! Whas enabled rapid disruption of virtually any locus of the human genome within one month,* g, m+ r* h0 s5 v9 k1 f" r F
and homozygous knockout clones lacking a human gene of interest can be created within 2–3) G- \0 R' u3 g3 u. d
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly: R T3 S- c* K
human—genes.7 [. m& `* L- u' A; N3 V
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发表于 2009-3-20 10:16
发表于 2009-4-9 12:06
好东东
