Abstract
The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.
Highlights
Genetic modification of mammalian genomes is of great importance for bio-medical researches such as deciphering gene functions, investigating disease mechanisms and searching and validating therapeutic targets, and a potential method to generate farm animals with improved economic traits for agricultural purposes.Mammalian genetic modification includes transgenesis, gene disruption and random mutation of genomes
With the development of powerful site-specific engineered endonucleases(EENs), especially Zinc Finger Nucleases(ZFNs) [1,2,3,4], Transcription Activator-like Effector Nucleases (TALENs)[5,6,7,8,9,10] and Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated system 9 (CRISPR/Cas9) [11,12,13,14,15], which are capable of disrupting genes efficiently by making double strand breaks (DSBs) at target sites, gene disruption has become a much more efficient and convenient process which is independent on embryonic stem cells (ESCs) and achieved significant success in mammalian species other than mice
The NLS-I-SceI molecule and transgene construct The NLS-I-SceI molecule consists of 36SV40 NLS, an HA tag epitope and the native I-SceI molecule as shown in Fig. 1 B. 36SV40 NLS is highly potent for nuclear localization, and HA tag epitope can be used to detect NLS-I-SceI molecule distribution in cells once it was expressed in cytoplasm
Summary
Mammalian genetic modification includes transgenesis, gene disruption and random mutation of genomes. Gene disruption was once a sophisticated and labor-intensive process which was based on DNA homologous recombination (HR) in embryonic stem cells (ESCs). This DNA HR-based technology achieved very limited success in mammalian species other than mice due to the lack of ESCs derived from these species. Mammalian transgenesis, especially for species other than mice, remains to be further optimized
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