Cerbini T, Funahashi R, Luo Y, Liu C, Park K, Rao M, et al. (2015) Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBLTargeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines. PLoS ONE 10(1): e0116032. doi:10.1371/journal.pone.0116032
Summary: The focus of this paper was to investigate targeted gene insertion using TALENs as a means of creating an efficient, long-lasting reporter stem cell line. Stem cell transfection is typically challenging and results in low percentage efficiencies. Here, they seek to increase transfection efficiencies by using TALENs to target reporter genes to two “safe harbor” sites (AAVS1 and CLYBL), either separately or both at the same time. They compared efficiencies in transient reporter expression as well as long-term expression in both neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). They found that CLYBL-targeted transgenes resulted in higher transgene expression and resulted in clones that stably express the transgene through division and differentiation into cardiomyocytes and neurons. They also were able to knock-in transgenes at dual safe harbor sites. Finally, transgene expression in the CLYBL gene does not greatly disrupt normal local and global gene expression. These data offer support for a gene that can be targeted for safe harbor integration to maintain long-term expression of reporter genes in stem cell lines.
Basic Methods: They used TALENs to target transgenes expressing reporters (tdTomato, GFP, Nanoluc-Halotag) into either the CLYBL locus or the AAVS1 site, both considered to be safe harbor sites. After transfection of TALENs and reporter plasmids into NSCs or iPSCS using the 4D-Nucleofector X Unit, cells were assessed for transgene expression by staining with Oregon green and assessing Nanoluc expression or looking at expression levels of tdTomato and GFP. They allowed stem cells to differentiate into cardiomyocytes or neurons and again assessed reporter expression. They evaluated the location of transgene integration by using Southern blot analysis. Finally, they investigated local and global gene expression via microarray and RT-qPCR.
Why It’s Important: We are interested in methods to achieve stable expression of reporter genes in human embryonic stem cells that can be inserted into the inner ear, ultimately differentiating into hair cells. In conjunction with the Raphael lab, we have previously established a method that can insert non-stem cells into the inner ear with some success. The next step is insertion of stem cells, and using these cells (with successfully integrated reporter genes) would allow us to further investigate insertion, differentiation, and viability of cells after transplantation into the inner ear.
PDF of paper: Cerbini et al 2015
Presentation ppt: Cerbini et al lab meeting 1.23.15