TY - JOUR
T1 - Targeted mutagenesis in Arabidopsis using zinc-finger nucleases.
AU - Zhang, Feng
AU - Voytas, Daniel F.
PY - 2011
Y1 - 2011
N2 - We report here an efficient method for making targeted mutations in Arabidopsis thaliana genes. The approach uses zinc-finger nucleases (ZFNs) - enzymes engineered to create DNA double-strand breaks at specific target loci. Imprecise repair of double-strand breaks by nonhomologous end-joining generates small insertions or deletions at the cleavage site. In this protocol, constructs encoding ZFNs for specific loci are transformed into Arabidopsis by Agrobacterium-mediated transformation. ZFN expression is induced during germination to initiate mutagenesis of the target locus. Typically, more than 20% of the primary transgenics segregate loss-of-function mutations in the next generation. ZFNs make it possible to expand the range of Arabidopsis mutants available for study and to create mutations in genes missed by random mutagenesis approaches, such as those using T-DNA, transposons, or chemical mutagens.
AB - We report here an efficient method for making targeted mutations in Arabidopsis thaliana genes. The approach uses zinc-finger nucleases (ZFNs) - enzymes engineered to create DNA double-strand breaks at specific target loci. Imprecise repair of double-strand breaks by nonhomologous end-joining generates small insertions or deletions at the cleavage site. In this protocol, constructs encoding ZFNs for specific loci are transformed into Arabidopsis by Agrobacterium-mediated transformation. ZFN expression is induced during germination to initiate mutagenesis of the target locus. Typically, more than 20% of the primary transgenics segregate loss-of-function mutations in the next generation. ZFNs make it possible to expand the range of Arabidopsis mutants available for study and to create mutations in genes missed by random mutagenesis approaches, such as those using T-DNA, transposons, or chemical mutagens.
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U2 - 10.1007/978-1-61737-957-4_9
DO - 10.1007/978-1-61737-957-4_9
M3 - Article
C2 - 21181530
AN - SCOPUS:79953783824
SN - 1064-3745
VL - 701
SP - 167
EP - 177
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -