A transgenic-cloned pig model expressing non-fluorescent modified Plum.

Genetically modified pigs that express fluorescent proteins such as green and red fluorescent proteins have become indispensable biomedical research tools in recent years. Cell or tissue transplantation studies using fluorescent markers should be conducted, wherein the xeno-antigenicity of the fluorescent proteins does not affect engraftment or graft survival. Thus, we aimed to create a transgenic (Tg)-cloned pig that was immunologically tolerant to fluorescent protein antigens. In the present study, we generated a Tg-cloned pig harboring a derivative of Plum modified by a single amino acid substitution in the chromophore. The cells and tissues of this Tg-cloned pig expressing the modified Plum (mPlum) did not fluoresce. However, western blot and immunohistochemistry analyses clearly showed that the mPlum had the same antigenicity as Plum. Thus, we have obtained primary proof of principle for creating a cloned pig that is immunologically tolerant to fluorescent protein antigens.

IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis.

The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53-dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future.

A new system to evaluate the influence of immunosuppressive drugs on pancreatic islets using epigenetic analysis in a 3-dimensional culture.

OBJECTIVE: The present study aimed to establish a new method to evaluate the influence of immunosuppressive drugs on pancreatic islets in short-term in vitro cultures using epigenetic analysis in a 3-dimensional culture. METHODS: For this purpose, we selected (a) a 3-dimensional culture system utilizing thermoreversible gelation polymer, (b) pancreatic duodenal homeobox-1 (pdx-1)-Venus transgenic pigs expressing the green fluorescent protein, (c) FK506 as an immunosuppressive drug of the evaluation, and (d) the bisulfite sequencing technique to evaluate the methylation levels of pdx-1 and insulin genes. Each isolated pancreatic islet was cultured with several doses of FK506. The viability of the each islet was evaluated by analyzing the emission of Venus in real time and by propidium iodide staining. Epigenetic analysis was performed at several time points. RESULTS: Each single pancreatic islet was stably cultured for 30 days in this system. At day 30 in culture, we observed that insulin DNA methylation levels in the group that received a high dose of FK506 dramatically increased, although there was no change in pdx-1 DNA methylation level and configuration of the islets. CONCLUSIONS: Our system may be useful to determine immunosuppressive drugs that are specifically suitable for islet transplantation.

Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).

Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.

Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA.

Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.