Production of cloned embryos using PRNP-knockout fibroblasts

Abstract

Prion is one of the membrane protein of most kinds of cells, but its conformational changes and accumulations induce transmissible spongiform encephalopathies (TSE), known as mad cow disease, in mammalian species. The first production of prion-free cattle was reported in 2007 by homologous recombination, but a wide range of physiological and pathological functions of prion is still unknown, and the gene targeting using homologous recombination is time and cost consuming process. Recently, the powerful nucleases, transcription activator-like effector nuclease (TALEN) and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9, have been widely used for site-specific DNA recombination. They have a wide range of applicability from plants to mammalians, and the efficiency was known to be high. In this study, the prion protein (PRNP)-disrupted bovine cell lines have been established which are readily applicable to generate PRNP-free embryos, and functional studies on prion were conducted. For the first step of gene targeting in bovine cells, immortalization of bovine fibroblasts was tried to enhance efficiency of in vitro study. Two genes, B lymphoma moloney murine leukemia virus (Mo-MLV) insertion region 1 homolog (BMI1) and human telomerase reverse transcriptase (hTERT), were introduced into bovine fibroblasts by random integration, and proliferation of the BMI1+hTERT introduced cells was maintained after 60 passage in vitro, although the naïve fibroblasts and the BMI1-introduced cells were arrested after 20 and 40 passages, respectively. In the reconstructed embryos via somatic cell nuclear transfer (SCNT), the immortalized cells were successfully reprogramed, and the embryos were developed up to preimplantation stages. The expression level of p53, one of tumor suppressor, was maintained at low in the whole period of in vitro culture, and the telomerase activity was significantly increased at passage 60 compared with passage 20. These expression alterations compared with other cell lines are considered as supporting factors for cell proliferation in the immortalized cells. Nextly, customized TALEN was designed to eliminate PRNP expression. The TALEN pair targeting exon 3 of PRNP gene and the reporter-expressing vectors were transferred into the immortalized cells, first. By co-transfection with the reporter vector, it was capable of enrichment of mutation–positive cells. After sorting the transfected cells, they were seeded to generate monoclonal cell population, and each cell populations were collected and analyzed. Among the isolated cell lines, about 67 % of cell lines were T7E1 positive and about 19 % were biallelic mutations. In the sequence analysis, various types of mutations including deletions and insertions were observed, and the mutation patterns were consistently maintained in the cloned embryos. Although the mRNA expression of PRNP was not observed in the PRNP-knockout embryos, they were fully developed until blastocysts stage with similar developmental rate with controls. Expression analysis of the PRNP-related genes was opposed to the previous study, and further investigation should be needed to reveal the gene expressions according to the cell line and mutation type. The normal type of prion expression was also evaluated in different bovine organs and ovarian follicular structures according to estrus cycles. As expected, the prion expression was strong in spinal cord compared with the expressions in others (heart, liver, lung, spleen, kidney, and ovary). In the ovarian follicles, the prion expression altered depending on follicular stages. All kinds of corpus luteum expressed prion strongly, but the expression was weakly detected in the dominant follicles larger than 8 mm of diameter. In addition to the PRN-knockout experiments, the PRNP-targeting knockin vector containing homology arms of the TALEN domains was successfully integrated into bovine genomic DNA. The integration of exogenous sequence was confirmed through PCR analysis, and the mRNA expression of PRNP was eliminated by the result of the knockin mutation targeting PRNP sequence. Following successful knockout and knockin via TALEN in bovine cells, genomic mutation using CRISPR/Cas9 system was induced in the bovine cells. As a screening research of CRISPR/Cas9 system on bovine cells, knockout of exogenous DNA was induced. To evaluate the knockout efficiency of CRISPR/Cas9, the enhanced green fluorescent protein (eGFP)-expressing cell line was established: the piggyBac-mediated eGFP integration was induced in bovine fibroblasts, and its cloned embryo was transferred to collect transgenic fetus. The green fluorescence expression was confirmed under fluorescence microscope. The CRISPR/Cas9 expressing vectors targeting exogenous eGFP sequence were transferred into the eGFP fetal fibroblasts by single electroporation stimuli, and about 40 % of eGFP cells have lost their fluorescence signals. The disruption of exogenous gene did not affect to the capability of knockout cells to generate blastocysts in vitro. Finally, the CRISPR/Cas9 targeting bovine PRNP gene was designed and transfected with the reporter vector. The PRNP mutation rates induced by CRISPR/Cas9 were 66 % and 70 % among the monoclonal colonies of the immortalized cells. Moreover, more than a half of the monoclonal cell lines were mutation-positive also in the normal bovine fibroblasts. The mutation patterns were confirmed through sequence analysis and fluorescent PCR (fPCR), and they were consistent in the cloned embryos generated by SCNT. After transfer the PRNP-mutant cloned embryo into the recipient, successful implantation was confirmed at day 40 through ultrasound imaging. In conclusion, the bovine immortalized cells with exogenous BMI1 and hTERT were stably established and applied for bovine in vitro studies. In addition to that, mutations on PRNP were effectively induced by introduction of TALEN and CRISPR/Cas9, respectively, and PRN-knockout embryos were developed normally in vitro despite a lack of understanding of prion. These genetic tools are expected to be applicable to generate transgenic animals which have a relatively large diversity of targeted mutations.