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Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

ABSTRACT: BACKGROUND:Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been demonstrated that these programmable nucleases can introduce genetic changes in embryos from many species including NHPs. In 2014, we reported the first genetic editing of macaques using TALENs and CRISPR/Cas9. Subsequently, we characterized the phenotype of a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model of Rett syndrome generated using the TALEN approach. These efforts not only accelerated the advance of modeling genetic diseases in NHPs, but also encouraged us to develop specific gene knock-in monkeys. In this study, we assess the possibility of homologous recombination (HR)-mediated gene replacement using TALENs in monkeys, and generate preimplantation embryos carrying an EmGFP fluorescent reporter constructed in the OCT4 gene. RESULT:We assembled a pair of TALENs specific to the first exon of the OCT4 gene and constructed a donor vector consisting of the homology arms cloned from the monkey genome DNA, flanking an EmGFP cassette. Next, we co-injected the TALENs-coding plasmid and donor plasmid into the cytoplasm of 122 zygotes 6-8?h after fertilization. Sequencing and immunofluorescence revealed that the OCT4-EmGFP knock-in allele had been successfully generated by TALENs-mediated HR at an efficiency of 11.3% (7 out of 62) or 11.1% (1 out of 9), respectively, in monkey embryos. CONCLUSION:We have successfully, for the first time, obtained OCT4-EmGFP knock-in monkey embryos via HR mediated by TALENs. Our results suggest that gene targeting through TALEN-assisted HR is a useful approach to introduce precise genetic modification in NHPs.

SUBMITTER: Chu C

PROVIDER: S-EPMC6334428 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

Chu Chu C Yang Zhaohui Z Yang Jiayin J Yan Li L Si Chenyang C Kang Yu Y Chen Zhenzhen Z Chen Yongchang Y Ji Weizhi W Niu Yuyu Y

BMC biotechnology 20190115 1

<h4>Background</h4>Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been ...[more]

PMID: 30646876

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Project description:Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-?, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 ?g/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6-8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

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Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

Publications

Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

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