GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

Area of Science:

• Stem cell biology
• Molecular biology
• Genetic engineering

Background:

• Reliable gene expression is crucial for studying human pluripotent stem cells (hPSCs).
• Investigating cell fates and gene function in hPSCs requires robust reporter systems.

Purpose of the Study:

• To develop and validate a simple, reliable gene expression system for hPSCs.
• To enable indelible labeling of hPSCs and their differentiated progeny.

Main Methods:

• Insertion of reporter genes (GFP, mCherry, luc2, lacZ, etc.) into the GAPDH locus of hPSCs using GAPTrap (GT) vectors.
• Analysis of reporter gene expression consistency in independent clones.
• Assessment of reporter gene stability during differentiation into various cell types (hematopoietic, cardiac, endoderm, neurons) and in teratomas.

Main Results:

• GAPTrap vectors demonstrated remarkably consistent reporter gene expression levels across independent hPSC clones.
• Reporter expression was reliably maintained in differentiated cells, including hematopoietic cells, cardiac mesoderm, definitive endoderm, and dopaminergic neurons.
• Analysis of teratomas confirmed stable β-galactosidase expression in derivatives from all three germ layers.

Conclusions:

• The GAPTrap system provides a robust and straightforward method for indelible labeling of hPSCs.
• This system ensures consistent reporter gene expression, facilitating research on cell fates and gene function in stem cell derivatives.