The evolutionary fate of rpl32 and rps16 losses in the Euphorbia schimperi (Euphorbiaceae) plastome

Gene transfers from mitochondria and plastids to the nucleus are an important process in the evolution of the eukaryotic cell. Plastid (pt) gene losses have been documented in multiple angiosperm lineages and are often associated with functional transfers to the nucleus or substitutions by duplicated nuclear genes targeted to both the plastid and mitochondrion. The plastid genome sequence of Euphorbia schimperi was assembled and three major genomic changes were detected, the complete loss of rpl32 and pseudogenization of rps16 and infA. The nuclear transcriptome of E. schimperi was sequenced to investigate the transfer/substitution of the rpl32 and rps16 genes to the nucleus. Transfer of plastid-encoded rpl32 to the nucleus was identified previously in three families of Malpighiales, Rhizophoraceae, Salicaceae and Passifloraceae. An E. schimperi transcript of pt SOD-1-RPL32 confirmed that the transfer in Euphorbiaceae is similar to other Malpighiales indicating that it occurred early in the divergence of the order. Ribosomal protein S16 (rps16) is encoded in the plastome in most angiosperms but not in Salicaceae and Passifloraceae. Substitution of the E. schimperi pt rps16 was likely due to a duplication of nuclear-encoded mitochondrial-targeted rps16 resulting in copies dually targeted to the mitochondrion and plastid. Sequences of RPS16-1 and RPS16-2 in the three families of Malpighiales (Salicaceae, Passifloraceae and Euphorbiaceae) have high sequence identity suggesting that the substitution event dates to the early divergence within Malpighiales.

The evolutionary fate of rpl32 and rps16 losses in the Euphorbia schimperi (Euphorbiaceae) plastome

Gene transfers from mitochondria and plastids to the nucleus are an important process in the evolution of the eukaryotic cell. Plastid (pt) gene losses have been documented in multiple angiosperm lineages and are often associated with functional transfers to the nucleus or substitutions by duplicated nuclear genes targeted to both the plastid and mitochondrion. The plastid genome sequence of Euphorbia schimperi was completed and losses of rpl32, rps16 and infA genes were detected. The nuclear transcriptome of E. schimperi was sequenced to investigate the transfer/substitution of the rpl32 and rps16 genes to the nucleus. Transfer of plastid-encoded rpl32 to the nucleus was identified previously in three families of Malpighiales, Rhizophoraceae, Salicaceae and Passifloraceae. An E. schimperi transcript of pt SOD-1-RPL32 confirmed that the transfer in Euphorbiaceae is similar to other Malpighiales indicating that it occurred early in the divergence of the order. Ribosomal protein S16 (rps16) is encoded in the plastome in most angiosperms but not in Salicaceae and Passifloraceae. Substitution of the E. schimperi pt rps16 was likely due to a duplication of nuclear-encoded mitochondrial-targeted rps16 resulting in copies dually targeted to the mitochondrion and plastid. Sequences of RPS16-1 and RPS16-2 in the three families of Malpighiales (Salicaceae, Passifloraceae and Euphorbiaceae) have high sequence identity suggesting that the substitution event dates to the early divergence of Malpighiales.

Frameshift mutation in myostatin gene by zinc-finger nucleases results in a significant increase in muscle mass in Meishan sows

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. A significant increase in skeletal muscle was observed in Mstn^−/− mice compared with wild-type mice. So far, there has been no report on porcine MSTN mutations leading to skeletal muscle hypertrophy. In this report a MSTN frameshift mutation missing 11 nucleotides in exon 2 was introduced into Meishan pigs by zinc finger nuclease (ZFN) technology. ZFN-edited MSTN^−/− Meishan pigs were successfully produced by a cloning method of somatic cell nucleus transfer. Results from slaughter experiments indicated that lean meat yield increased 16.53% in about 80 kg (10-months-old) MSTN^−/− Meishan sows compared with their corresponding wild-type counterparts. The lean percentage of carcass from MSTN^−/− sows was 61.20% vs 48.25% for MSTN^+/− sows and 44.67% for wild-type sows. The fat of MSTN^−/− sows was significantly lower than that of MSTN^+/− and wild-type sows. The loin eye area of MSTN^−/− Meishan sows (56.42 cm²) was greater than that of MSTN^+/− (37.39 cm²) and wild-type (26.26 cm²) sows. The muscle fibre area of longissimus muscle in wild-type Meishan sows (1 946 μm²) was significantly greater than that of MSTN^+/− (1 324 μm²) and MSTN⁻^/− (1 419 μm²) sows. Moreover the significantly increased skeletal muscle in these MSTN^−/− Meishan sows was mainly due to the increase in the number of myofibres rather than to hypertrophy. Compared with wild-type Meishan sows, it was noted that myofibres had transformed from type I to IIB in MSTN^−/− Meishan sows. Our present study demonstrated that frameshift mutation in MSTN by ZFN technology led to a significant increase in muscle mass and a significant decrease in fat content in Meishan sows.

208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)

Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P<0.05) was induced, and reprogrammed fibroblasts were differentiated into chondrogenic and osteogenic lineages. Immunohistochemistry positivity for OCT4 was detected in treatment II. In summary, we showed that dermal fibroblasts of pampas cat can be reprogrammed into cells with multipotent characteristics, particularly when a cocktail of 5-azacytidine + VitC + A8301 + PDFG was used. Treatment I probably failed because of other growth factors and proteins present in PRP, which might inhibit successful reprogramming or activate other pathways leading to a nonmultipotent phenotype. Further refinements of these protocols are required to improve the reprogramming protocol. This in turn should help us obtain cells that can be used in nucleus transfer or cellular therapies in endangered felid species.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.