cDNA

pitx2_L (OZ533)

27210

pitx2_L (OZ533)

Kanamycin

This plasmid encodes a zinc finger array targeting half of a target sequence in the zebrafish gene pitx2 (see below). Please note that this plasmid does NOT contain the pitx2 sequence. Users must order the complementary plasmid pitx2_R (OZ534) [Addgene plasmid 27211] in order to create a zinc finger nuclease (ZFN) pair to introduce targeted mutations into this specific zebrafish gene. Users will also need to clone the zinc finger (ZF) insert of this plasmid into a zinc finger nuclease (ZFN) vector to express a FOKI fusion product. Examples of possible ZFN expression vectors that can be used are: pST1374 (Addgene plasmid 13426), pMLM290 (Addgene plasmid 21872), pMLM292 (Addgene plasmid 21873), pMLM800 (Addgene plasmid 27202) and pMLM802 (Addgene plasmid 27203). This zinc finger array was tested for binding activity to the sequence 5'-GCTGAAGCG-3' in a bacterial two hybrid assay, and resulted in 8.81 fold activation. However, this array has not yet been tested for activity as a zinc finger nuclease (i.e. for its ability to induce mutations at the intended locus). Scientists using this zinc finger array in a publication should notify zebrafishzfs@zincfingers.org and acknowledge NIH grant number R01 GM088040 in the publication. Other Articles: Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays. Maeder ML et al. (Nat Protoc. 2009 Sept 17. 4(10):1471-1501. Pubmed ID: 19798082 Targeted mutagenesis in zebrafish using customized zinc-finger nucleases. Foley JE et al. (Nat Protoc. 2009 Dec 3. 4(12):1855-1867. Pubmed ID: 20010934

Requires a bacterial strain such as XL1 Blue, that expresses the lacIq allele of lac repressor

37

Low Copy