In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis-or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.apancreatic pig | organ reconstitution | transplantation | somatic cell nuclear transfer | chimera

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Two experiments were conducted to investigate the effect of dietary taurine and cystine on growth and body composition of juvenile red sea bream Pagrus major. In Experiment I, a casein-based semi-purified diet included a small amount of fish meal were supplemented with taurine at the levels of 0 (control) and 1.0 %. The experimental diets in Experiment II were without fishmeal and supplemented with taurine at 0 (control), 0.5, 1.0 and 2.0 % or cystine at 1.0 and 2.0%. These diets were fed three times a day for 6 weeks to fish (average body weight: 2.3g in Experiment I and 2.5g in Experiment II). In Experiment I, fish fed the taurine supplemented diet showed significantly (P<0.05) improved growth, feed efficiency and feed consumption relative to fish fed the unsupplemental diet. The whole body taurine content increased, whereas the non-essential amino acid contents decreased, in fish fed the taurine-supplemental diet compared to fish fed the unsupplemented diet. In Experiment II, the growth, feed efficiency and feed consumption of fish fed the taurine-supplmented diets, irrespective of the dietary taurine levels, were significantly higher than those of fish fed the control diet and the cystine-supplemented diets.Taurine content in the whole body increased with the dietary taurine level, while the taurine contents did not increase by the supplemental cystine. Other free amino acid contents in the taurine supplemented diet groups followed similar trends to those in Experiment I. These results indicate that supplemental taurine to a casein-based semi-purified diet at more than 0.5% improved the growth and feed performance of juvenile red sea bream. It is also suggested that juvenile red sea bream can not metabolize cystine into taurine.3

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.

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There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal's ureter to the cloacaldeveloped bladder, a technique we called the "stepwise peristaltic ureter" (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.cloned pig | kidney generation | metanephros | somatic cell nuclear transfer | transplantation

The effect of taurine on growth of yellowtail juveniles Seriola quinqueradiata was investigated by a feeding experiment of diets containing various taurine levels. Test diets supplemented with 0, 0.5, 1.0, 1.5 and 2.0% of taurine were prepared. These diets were fed to yellowtail juveniles with an initial mean body weight of 0.5 g for 6 weeks. Supplementation of taurine in the diet of yellowtail improved their growth performance significantly (P < 0.05) over the initial 3-week period. The fish fed with the taurine-supplemented diet improved in percent gain and feed efficiency over both 3 and 6 weeks. Taurine content in the muscle proportionally increased with the dietary taurine level. The fish fed without supplemented taurine diet showed higher contents of serine in the muscle. With each increase in the inclusion level of taurine content in the diet, the concentration of serine in the muscle decreased. The cystathionine content in the muscle of each group was unchanged. These results suggest that taurine supplementation in the diet not only improves growth but also affects the sulfur amino acid metabolism of yellowtail juveniles.