Suitability of a universal electroporation device for genome editing and production of transgenic rats.

Abstract

Mammalian genome editing has utilized expensive and highly specialized electroporator devices. The "Gene Pulser XCell," a modular electroporation system for transfecting all cell types, has not been used extensively in mammalian embryo genome editing. The present experiment was undertaken to determine the usefulness of the Gene Pulser XCell for inserting the CRISPR/Cas9 system into intact zygotes in order to obtain the enhanced green fluorescent protein reporter rats (eGFP-R). An electroporation pulse response test using mCherry mRNA was performed to optimize the settings of the electroporator. Forty-five combinations of five pulse voltages (15, 25, 30, 35 and 40V), three pulse durations (5, 10 and 25ms), and three pulse frequencies (2, 5 and 6 pulses) applied at a constant 100-ms pulse interval and temperature of 37.5°C were evaluated. The test revealed that the 35V was the only voltage suitable for insertion of mCherry mRNA into intact rat zygotes and the only one that resulted in the production of embryos attaining the blastocyst stage. The incorporation of mCherry mRNA increased but the survival of the electroporated embryos declined with an increment in the number of pulses. Subsequent transfer of 1112 surviving Sprague Dawley rat embryos (after 8h of incubating 1800 zygotes electroporated with the CRISPR/Cas9) resulted in the production of 287 offspring (25.8%). Ensuing PCR and phenotypic evaluation confirmed that twenty animals (6.96%) expressed eGFP in all body organs/tissues except for blood and blood vessels. The mortality of males and females before the attainment of puberty was 2 and 3 pups, respectively, and the final number/ratio of male to female of offspring was 9:11. All the surviving rats mated naturally and successfully transmitted the GFP transgene to their progeny. The Gene Pulser XCell total system with the settings predetermined in the present experiment can effectively be used to produce transgenic rats through the CRISPR/Cas9-mediated genome editing of zygotes.