Nucleofection Mediates High-efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

Authors:
Hohenstein KA, Pyle AD, Chern JY, Lock LF, Donovan PJ
In:
Source: Stem Cells
Publication Date: (2008)
Issue: 26(6): 1436-43
Research Area:
Stem Cells
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Platform:
Nucleofector™ I/II/2b
Experiment
Optimization of Nucleofection: six programs (A-006, A-012, A-013, A-023, A-027, B-016), two solutions, and two cell harvesting methods (collagenase, trypsin) were tested. Best results were obtained by using trypsin-derived single cells with program A-023 (transfection efficiency 61 %), and these conditions were used in all following experiments. 2x10^6 hES cells (H1 or H9 cell lines) were trypsinized, resuspended in 100 µL mES solution plus 2-3 µg DNA (or shRNA, siRNA). Nucleofection of pmaxGFP or 4.9kb vector containg hrGFP (GFP-neo) resulted in efficiencies with a mean of 76 %. Stable GFP-neo expression (selection by G418) achieved 85 % efficiency. RNAi: Transfection of shRNA expressing vector or siRNA (co-transfected with pmaxGFP) against Oct 4 or Nanog; analysis by RT-PCR with beta-2M gene as control. Data strongly support that nucleofection of siRNA/shRNS vectors effectively alters gene expression. Stable introduction of AP-specific shRNA reduces expression of the gene without effecting hES cell characteristics. Altogether, using nucleofection combined with culture conditions improving ES cell survival, transient transfection efficiencies of up to 85 % and a stable transfection efficiency of up to 1.2 in 10^4 cells was achieved.
Abstract
High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient, but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Further, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed hrGFP. These clones retained stem cell characteristics; normal karyotype, stem cell marker expression, self-renewal, and pluripotency. These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells.