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. 2011 Jul 7;29(8):731-4.
doi: 10.1038/nbt.1927.

Genetic engineering of human pluripotent cells using TALE nucleases

Dirk Hockemeyer  1 , Haoyi Wang, Samira Kiani, Christine S Lai, Qing Gao, John P Cassady, Gregory J Cost, Lei Zhang, Yolanda Santiago, Jeffrey C Miller, Bryan Zeitler, Jennifer M Cherone, Xiangdong Meng, Sarah J Hinkley, Edward J Rebar, Philip D Gregory, Fyodor D Urnov, Rudolf Jaenisch
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Genetic engineering of human pluripotent cells using TALE nucleases

Dirk Hockemeyer et al. Nat Biotechnol. .
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Abstract

Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

Figures

Figure 1. Genetic engineering of hESCs and iPSCs using TALENs

Schematic overview depicting the targeting strategy for the PPP1R12C gene. Southern blot probes are shown as red boxes, exons as blue boxes; the arrow indicates cut site by the TALENs. Donor plasmids: SA-2A-Puro - splice acceptor sequence followed by a 2A self-cleaving peptide sequence and the puromycin resistance gene; pA - polyadenylation sequence;PGK - phosphoglycerate kinase promoter; Puro - puromycin resistance gene; CAGGS - synthetic CAGGS promoter–containing the actin enhancer and the CMV early promoter; eGFP - enhanced green fluorescent protein. Below, scheme of PPP1R12C TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable di-residue (RVD); each RVD is related to the cognate targeted DNA base by the following code (NI = A, HD = C, NN = G, NG = T).

Southern blot analysis of WIBR#3 hESCs targeted using PPP1R12C TALENs and the SA-2A-Puro donor plasmid. Genomic DNA was digested with SphI and hybridized with an 32P-labeled external 3’-probe (Top) or with an internal 5’-probe (bottom). SA-2A-Puro 5’-probe detects a 6.5 kb wt and a 3.8 kb trageted fragment; 3’-probe a 6.5 kb wt and a 3.7kb targeted fragment. Wt = wild type and T = correctly targeted allele.

Schematic overview depicting the targeting strategy for the OCT4 locus using the OCT4-STOP TALENs. Southern blot probes and exon of OCT4 are colored as in panel A and the vertical arrow indicates the OCT4-STOP TALEN cut site. Shown above are the donor plasmid used to target the OCT4 locus, loxP-sites are shown as red triangles, UTR: untranslated region of the OCT4 gene.

Southern blot analysis of the WIBR#3 hESCs targeted in the OCT4 locus with the OCT4-eGFP-PGK-Puro donor plasmids. Genomic DNA was digested with BamHI and hybridized with the 32P-labeled external 3’-probe or with the internal eGFP probe. Two targeted clones are shown, one correctly targeted and one carrying a random integration. Expected fragment size: wt=4.2kb, targeted=6.8kb for both probes. Left lane: wt clone; middle lanes: clones before and after excision (right lane) of PGK-Puro cassette.

Southern blot analysis as in (D) of WIBR#3 hESCs targeted with 2A-eGFP-PGK-Puro donor plasmids. Expected fragment size as in (D). A clone before (left lane) and two clones after (right lanes) Cre-mediated excision of PGK-Puro cassette.

Southern blot analysis as in (D and E) of WIBR#3 hESCs targeted with the eGFP-2A-Puro donor plasmids. Expected fragment size: wt 4.2kb, targeted 5.6kb for both probes. Two correctly targeted clones (left two lanes) and one targeted clone carriying additional aberrant integration (right lane).

Left and middle panels show phase contrast images (top row) and corresponding eGFP fluorescence (bottom row) of OCT4-eGFP and OCT4-2A-eGFP hESCs after excision of the PGK-Puro cassette at two magnifications. Right panels: phase contrast images and eGFP fluorescence of OCT4-eGFP-2A-Puro targeted hESCs clones. Size bars 100µm.

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References

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