Generation of Genetically Modified Mice by Oocyte Injection of Androgenetic Haploid Embryonic Stem Cells

SummaryHaploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.PaperCli

Transient exposure to miR-203 expands the differentiation capacity of pluripotent stem cells

Full differentiation potential along with self‐renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro . We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already‐established murine and human PSCs. Short exposure to miR‐203 in PSCs (mi PSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human–mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR‐203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine

Engineering of glycerol utilization in Gluconobacter oxydans 621H for biocatalyst preparation in a low-cost way

Abstract Background Whole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications. Results G. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ∆GOX1068∆GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ∆GOX1068∆GOX0854 as the biocatalyst, 61.6 g L−1 xylonate was produced from 58.4 g L−1 xylose at a yield of 1.05 g g−1. Conclusion This process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol

Enzymatic Cascades for Efficient Biotransformation of Racemic Lactate Derived from Corn Steep Water

The corn starch industry produces a large amount of corn steep water, leading to high organic waste load. Lactate could be separated from corn steep water at a low cost, which supports the value-added utilization of corn steep water. However, the racemic characteristic of lactate from corn steep water restricts the development of this promising process. In this study, using d-lactate oxidase (d-LOX) from <i>Gluconobacter oxydans</i>, l-lactate oxidase (l-LOX) from <i>Pediococcus</i> sp., pyruvate decarboxylase from <i>Zymomonas mobilis</i>, and catalase from bovine liver, we synthesized an <i>in vitro</i> enzymatic system, including different enzymatic cascades, for the production of valuable platform chemicals from lactate separated from the corn steep water. Pyruvate was produced as an important intermediate and further converted into C2 (acetaldehyde) and C4 (acetoin) platform chemicals at high yields using optimized concentrations of pyruvate decarboxylase. The <i>in vitro</i> enzymatic system not only provides a novel technology platform for the production of optical lactate and lactate derivatives but also supports the sustainable development of corn starch industry

Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value