Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

Backgroundα1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation.MethodsTranscription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting.ResultsThe luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet.ConclusionsTALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0212-7) contains supplementary material, which is available to authorized users.

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48V Mild Hybrid vehicle has attracted more and more attention recently. Based on United Automobile Electronic Systems (UAES) 48V system, this paper presents us an optimized energy management strategy which mainly consists of operating mode analysis and torque distribution strategy. What's more, the fuel consumption was tested by UASE Labcar HIL bed in NEDC cycle. Consequently, the oil saving of 48V mild hybrid electrical vehicle can reach 9.24% with start/stop function.

With the development of China’s economy and the deepening of urbanization, the number of migrants whose parents have migrant work experience continues to rise. However, what is the long-term impact of parental migrant work experience on migrant children? Existing literature has not yet adequately answered. Based on the data from the China Migrants Dynamic Survey in 2016–2017, this article uses a multiple linear regression model to examine the impact of parental migrant work experience on the income of rural-urban migrants and its impact mechanism and heterogeneity empirically. We find that parental migrant work experience has a positive impact on the monthly income of second-generation rural-urban migrants. Specifically, compared with those whose parents had no such experience, the average monthly income of those whose parents had such experience increased significantly by 3.08% (approximately 124 yuan), and this effect was more apparent when fathers had migrant work experience. The main influencing channel comes from the significant increase in the probability of rural-urban migrants choosing self-employment. The results of the heterogeneity analysis showed that this effect was more significant in the sample of males and those with high school education and below. After a series of robustness tests, these conclusions remain valid. This work enriches the corresponding research literature and provides empirical evidence for studying the long-term effects of parents’ early experiences on their children.