cDNA

esrrga_L (OZ563)

33377

esrrga_L (OZ563)

Kanamycin

This plasmid encodes a zinc finger array targeting half of a target sequence in the zebrafish gene esrrga. Please note that this plasmid does NOT contain the esrrga sequence. Users must order the complementary plasmid esrrga_R (OZ564) [Addgene plasmid 33378] 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), pMLM290/292 (Addgene plasmids 21872 & 21873), pMLM800/802 (Addgene plasmids 27202 & 27203). The difference between pMLM800/802 and pMLM290/292 is the length of the spacer sequence in the full ZFN target site. If the spacer is 7 bp, scientists should use pMLM800/802. If the spacer is 5 or 6 bps, scientists should use pMLM290/292. pMLM290/292 is identical to pST1374 except that it harbors two mutations in the FokI nuclease domain (Q486E, I499L; aka the - mutation see Miller et al., Nat. Biotech 2007, PMID 17603475) which confers heterodimeric behavior on these domains This zinc finger array was tested for binding activity to the sequence 5'-GCGGTAGCT-3' in a bacterial two hybrid assay, and resulted in 4.39 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