Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

ATDC5: An excellent in vitro model cell line for skeletal development

The ATDC5 cell line is derived from mouse teratocarcinoma cells and characterized as a chondrogenic cell line which goes through a sequential process analogy to chondrocyte differentiation. Thus, it is regarded as a promising in vitro model to study the factors that influence cell behaviors during chondrogenesis. It also provides insights in exploring signaling pathways related to skeletal development as well as interactions with innovative materials. To date, over 200 studies have utilized ATDC5 to obtain lots of significant findings. In this review, we summarized the literature of ATDC5 related studies and emphasized the application of ATDC5 in chondrogenesis. In addition, the general introduction of ATDC5 including its derivation and characterization is covered in this article. J. Cell. Biochem. 114: 1223–1229, 2013. © 2012 Wiley Periodicals, Inc.     

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Microsatellite genetic variation in the Chinese endemic Eucommia ulmoides (Eucommiaceae): implications for conservation

Eucommia ulmoides, a traditional Chinese medicinal plant, is endangered as a consequence of long‐term and widespread harvest in the late 20th century. It has been widely cultivated as a source of herbal medicine and for use in the organic chemical industry in China. In this study, eight microsatellite markers were applied to investigate genetic diversity in E. ulmoides. Three hundred individuals from one semi‐wild population and nine cultivated populations across its main production area were collected. A high level of genetic diversity at population levels (H_E = 0.716) was observed. The highly outcrossed mating system, high longevity of E. ulmoides and seed admixture may be responsible for high genetic variation within populations. A genetic bottleneck was observed in one population. Populations were only slightly differentiated from one another (F_ST = 0.063); this was also supported by AMOVA, which revealed that 94.05% of the total variation resided within populations. This is probably attributable to long‐distance gene flow mediated by the exchange of seeds by local farmers. Implications of these results for the conservation of genetic resources of E. ulmoides are discussed. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 775–785.     

Ostericum atropurpureum sp. nov. (Apiaceae) from Zhejiang,China

Ostericum atropurpureum G. Y. Li, G. H. Xia & W. Y. Xie (Apiaceae, Apioideae) from Zhejiang, China, is described and illustrated. It is closely related to O. huadongense Z. H. Pan & X. H. Li and O. sieboldii (Miquel) Nakai, but differs in having leaves with 1.5–9.0 cm long petiole, linear bracteoles 6–12 mm long, 5–9 rays, 7–14‐flowered umbellules, dark purple petals, broadly winged dorsal and lateral fruit ribs, 1.0–1.5 mm broad, 3–6 vittae in each furrow and 4–8 on the commissure.     

A genome walking strategy for the identification of nucleotide sequences adjacent to known regions

To identify the transposon insertion sites in a soil actinomycete, Saccharopolyspora spinosa, a genome walking approach, termed SPTA-PCR, was developed. In SPTA-PCR, a simple procedure consisting of TA cloning and a high stringency PCR, following the single primer-mediated, randomly-primed PCR, can eliminate non-target DNA fragments and obtain target fragments specifically. Using SPTA-PCR, the DNA sequence adjacent to the highly conserved region of lectin coding gene in onion plant, Allium chinense, was also cloned.