citations

                    DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.
                

Authors:

Zhu F, Gamboa M, Farruggio AP, Hippenmeyer S, Tasic B, Schüle B, Chen-Tsai Y, Calos MP.

In:

Source: Proc Natl Acad Sci USA
Publication Date: (2014)
Issue: 42(5): e34

Research Area:

Stem Cells

Cells used in publication:

Embryonic Stem Cell (ES), human: Species: human; Tissue Origin: embryo
Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Platform:

4D-Nucleofector® X-Unit

Experiment

Platform: 4D- Nucleofector System Cells: hES + hiPSC Solution: P3 Cells/sample: 0.8-1.6x10e6 in 100 µl TALEN plasmids: 1 µg of each TALEN plasmid plus 8 µg donor plasmid or 4 µg for each plasmid encoding phiC31 and Bxb1 and 4 µg donor Plasmid Summary (by Lonza): The authors developed a new strategy for controlled targeted modification of human ES or iPSC called DICE (dual integrase cassette exchange). They combined TALEN-mediated homologous recombination with site-specific integrases. Throughout this 2-step process involving co-transfection of several plasmids they used the 4D-Nucleofector™ System. In the first step they performed a TALEN-mediated targeted insertion of a cassette containing att binding sites (specific for phiC31 or Bxb1 integrases) plus a GFP marker into a newly identified safe harbour locus on Chr H11. Then, in a second step, plasmids encoding phiC31 and Bxb1 integrases were transfected together with a new donor plasmid to exchange the GFP-containing cassette against another cassette containing either an mCherry marker or neural transcription factors. Their strategy offers complete control over content, direction and copy number of inserted genes with a specificity of 100%. It seems to allow for rapid, efficient and precise gene insertion in ESC and iPSC and is suited for repeated modifications of the same locus.

Abstract

To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson's disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.

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