pdf:Simple one-week method to construct Simple one-week method to construct gene

sunny

Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1,
0 E' Q) M- ~2 U/ c. h( z% xKei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama15 _2 R& W" B+ L" |, K3 P8 b4 w
1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima,# f; u2 j6 S* v: F5 Q0 w
Japan
" ^' E5 h4 |4 |9 L. Z; }7 pBioTechniques 41:311-316 (September 2006)+ h9 T7 n- L; {, b, Q/ c
doi 10.2144/0001122335 v$ s  ~: U5 S4 b: @
Targeted gene disruption is a powerful tool for studying gene function in cells and animals.
) i, O. o+ f( G! C' }$ `! R: [In addition, this technology includes a potential to correct disease-causing mutations.* E; r7 |8 c, h, z9 U
However, constructing targeting vectors is a laborious step in the gene-targeting strategy,
4 i' ~+ X5 F! p! K2 Z" Y9 Leven apart from the low efficiency of homologous recombination in mammals. Here, we introduce& v- d! c$ e0 D( C
a quick and simplified method to construct targeting vectors. This method is based1 T, F2 Z# u: Q# [4 ^$ g. q
on the commercially available MultiSite Gateway® technology. The sole critical step is to
( u4 Y+ @3 w! w6 T3 ddesign primers to PCR amplify genomic fragments for homologous DNA arms, after which
+ w! `% r2 e6 D) _  Rneither ligation reaction nor extensive restriction mapping is necessary at all. The method9 c& B9 \8 K1 U" [  s# v2 z) b, ~+ `$ y
therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms7 _- b; g( L, b( [$ Y: ?$ {
whose genome has been sequenced. Recently, we and others have shown that the human pre-
- k% T' l" q8 e! FB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified
# d& S( c. ^/ K2 c- p2 W' }vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line
* k, W" Y5 ?, _# @' w( _has enabled rapid disruption of virtually any locus of the human genome within one month,
& U5 n7 y+ X6 u* a7 \and homozygous knockout clones lacking a human gene of interest can be created within 2–3, n* j& D5 V& i' \
months. Thus, our system greatly facilitates reverse genetic studies of mammalian—particularly
: h$ ?/ _; q" o$ ehuman—genes.
" [4 Q9 O; B/ V' [  }0 A; \; N4 j( h$ G  H) Z4 `  _/ l

游客，如果你要查看本帖隐藏内容请回复