Generation of a fluorescent GFP reporter line in human induced pluripotent stem cells (hiPSCs) provides enormous potentials in both basic stem cell research and regenerative medicine. on chromosome 19 which is ubiquitously expressed. It is considered a “safe harbor” based on the CGP77675 CGP77675 observation that non-pathogenic adeno-associated virus 2 (AAV2) integrates at this site. Monoallelic disruption of the gene does not appear to have an adverse effect on the targeted cells (Ogata et al. 2003 Smith et al. 2008 Zou et al. 2011 The AAVS1 site has an open chromatin conformation structure since it presents FN1 a DNase I-hypersensitive site(Lamartina et al. 2000 CGP77675 This structure allows trans-acting factors access to perform recombination transcription replication and chromosome segregation(Gross and Garrard 1988 The open chromatin structure at the AAVS1 site is also associated with cis-acting insulators. Insulators are DNA structures that functionally maintain the gene expression by direct block of enhancers from affecting different promoter domains thus acting as boundaries to the surrounding heterochromatin that silences the genes located within (Burgess-Beusse et al. 2002 An insulator-like structure has been described at the AAVS1 site (Ogata et al. 2003 Indeed transgenes integrated in the AAVS1 site show stable and long term expression in a variety of cell types including hESCs and hiPSCs (Hockemeyer et al. 2011 Smith et al. 2008 For example AAVS1-EGFP expression in hESCs has been shown by both us and other investigators to be robust and persistent in long-term cell cultures. After lineage differentiation more than 90% of the differentiated cells still express EGFP and are able to maintain fluorescence even after in vivo transplanting into mice CGP77675 infraction heart for at least seven weeks (Smith et al. 2008 Thus the AAVS1 locus serves as a useful site for generation of fluorescent reporter cell lines in hiPSCs. Critical Parameters and Troubleshooting AAVS1-TALENs activity To integrate EGFP donor in AAVS1 locus in hiPSCs designed TALENs must efficiently cleave the targeted AAVS1 site to create DSB. Our AAVS1-TALENs target the same DNA sequence described in the previous report (Hockemeyer et al. 2011 but are encoded by a different TALE structure. The activities of these customized TALENs targeting the endogenous AAVS1 site will have an essential impact on efficiency of following transgene integration and need to be first determined. That is why we have described NHEJ assay as a first protocol because it measures TALEN cutting activity at endogenous locus. We have chosen human embryonic 293T cells as cell source since they are easy to culture and have much higher nucleofection efficiency (> 90%) relative to that of hiPSCs (10-50 %). Based on our experiences using this protocol cutting efficiency of TALENs should be at least 10 – 30% in 293T which will give a reasonable number of targeted colonies in hiPSCs. NHEJ can be also done in other human cells lines such as K562 or hiPSCs but results in different efficiencies (higher in K562 and lower in hiPSCs) CGP77675 Nucleofection efficiency By combination of Nucleofector? Programs and cell type-specific transfection solutions developed by Lonza Nucleofection? system provides a convenient and non-virus method to deliver nucleic acids such as plasmid into the nucleus directly and efficiently. This system in our hand has a very high cell survival (at least 90%) after 2 days of nucleofection in hiPSCs. However the transfection efficiency is around 10 – 50% depending on the hiPSC lines. In order to increase nucleofection efficiency we recommend working out optimal conditions using pMaxGFP control vector included in the Kit and your available hiPSC lines adjusting cell numbers amounts of plasmids and Neleofector? parameters before targeting experiments. Our experiences suggest that >30% nucleofection efficiency can significantly help successful targeting. Dosage of drug selection After 48 hr following the nucleofection the EGFP and puromycin-resistant genes contained in the EGFP donor are expressed in most of the transfected cells and positive selection should begin. The purpose of application of puromycin in cell cultures is to selectively allow the cells with targeted integration to survive since random integration-only.