Abstract
Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein.
Highlights
PiggyBac (PB) is a DNA transposon element that was first isolated from Trichoplusia ni[1,2] and has shown an efficient transposition ability in many species[3,4,5]
The zygotes were obtained from Sprague Dawley (SD) and Dark Agouti (DA) strain rats
The protocol of co-injecting PB and PBase-encoding mRNA into pronuclei was developed to improve the efficiency of producing transgenic animals and prevent re-transposition events, and can obtain average transformation frequencies of 80%25
Summary
PiggyBac (PB) is a DNA transposon element that was first isolated from Trichoplusia ni[1,2] and has shown an efficient transposition ability in many species[3,4,5]. Embryo manipulation techniques, including traditional pronuclear injection[15], ESC germline transmission[17] and haploid ESC intracytoplasmic injection[18], have been developed for the production of transgenic rats. These methods are difficult to perform and are time-consuming. The RFP and eGFP genes were able to insert into many sites of the rat genome and were expressed in various organs. This stable insertion by the PB system was passed to the generation through germline transmission. This study provided a novel method to produce fluorescent rats that can be used as a fluorescent ESC platform to target differentiation potential
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