Abstract
Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.
Highlights
Electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells
We developed an electroporation protocol using the square-wave pulsing program of 250 V for induced pluripotency stem (iPS) cells and 300 V for murine embryonic fibroblasts (MEF) cells, 2 pulses, each 10 ms length, and 10 s interval in 4 mm cuvettes (Fig. 1)
The electrotransfection efficiency of MEF cells was 30% higher in OptiMEM-GlutaMAX compared to the Bio-Rad medium (p value < 0.05)
Summary
Electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. We developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas[9] plasmid system. Abbreviations CRISPR Clustered regularly interspaced short palindromic repeats Cas[9] CRISPR-associated protein 9 gRNA Guide RNA iPS Induced pluripotent stem cell KO Knockout MEF Mouse embryonic fibroblast RNPs Ribonucleoprotein. The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas[9] (CRISPR-associated protein 9) nuclease system is a straight-forward, versatile, and highly efficient tool for genome editing of various organisms. Still there is a huge room to improve the electroporation efficiency to transfer exogenous DNAs into primary cells, for large plasmids[7,11]
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