Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neo^R in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neo^R was removed and Cre expressed transiently in GFP-positive colonies; excision of neo^R was confirmed by single-blastocyst PCR in recloned blastocysts, with neo^R-free fibroblast cells as donors. There was no difference (P > 0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neo^R-free or neo^R-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P > 0.05) from those of the control. In conclusion, we successfully excised neo^R from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.     

桂林小花苣苔离体快速繁殖技术

对抗结核植物桂林小花苣苔(Chiritopsis repanda var. guilinensis)进行离体培养与快速繁殖技术研究。结果表明: 桂林小花苣苔叶片外植体的最适初代诱导培养基为MS+0.5 mg·L^–16-BA+0.05 mg·L^–1IBA, pH8.0; 最适继代增殖培养基为 MS+0.1 mg·L^–16-BA+0.05 mg·L^–1IBA, pH6.0, 繁殖系数7.0/35天; 最适生根培养基为1/2MS+0.2 mg·L^–1NAA, pH6.0, 生根率为93.6%。模拟桂林小花苣苔自然生境, 在春季对生根试管苗进行大棚移栽, 成活率达90%。根据上述快繁技术, 理论上每株试管苗每年可繁殖桂林小花苣苔种苗46万株。     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

间断低氧对大鼠下丘脑超微结构及前增食欲素水平的影响

目的探讨睡眠中间断低氧对大鼠下丘脑前增食欲素及受体水平的影响以及下丘脑超微结构的变化。方法大鼠分成对照组、间断低氧组和持续低氧组,分别给予吸入空气,持续低氧和间断低氧气体,并在实验开始后1d、3d、1w和4w应用RT-PCR方法测定大鼠下丘脑前增食欲素及受体水平,分析其间的变化关系,电镜观察下丘脑的超微结构变化。结果与对照组和持续低氧组比较,间断低氧4w后大鼠下丘脑前增食欲素mRNA水平明显降低,受体水平升高,但在持续低氧和对照组之间无明显差异。在低氧后1d、3d、7d后大鼠下丘脑前增食欲素mRNA降低,受体水平升高,在4w后,持续低氧组则接近正常。急性持续低氧大鼠超微结构变化更严重,而慢性间断低氧变化更持久。结论慢性间断低氧可以引起下丘脑前增食欲素下降及受体水平升高,急性持续低氧也可引起上述变化,而慢性持续低氧未引起增食欲素改变;慢性间断低氧大鼠下丘脑超微结构表现为严重而持久的变化。     

Secondary succession of plant communities in a subtropical mountainous region of SW China

Since 1985, originally forested mountainous areas of China have been allowed to return to their natural state after years of exploitation including agriculture, development, and logging. The reforms began earlier in less accessible locations, so that today the successional process is more advanced there. The vegetation in Luquan, Qiongzhusi, and Xishan near Kunming, central Yunnan, exhibits, in a limited area, a range of stages of plant succession that are widely encountered throughout the broader region, and thus affords a special opportunity for a comprehensive study. We analyzed the successional sequence of these various plant communities. They ranged from pioneer coniferous and/or pioneer deciduous broad-leaved stands to pre-mature semi-humid evergreen broad-leaved stands, through mixed coniferous and broad-leaved or mixed deciduous and evergreen broad-leaved stands. The succession proceeded from pioneer coniferous Pinus and Keteleeria, and deciduous Platycarya and Alnus, to late-successional evergreen broad-leaved Cyclobalanopsis and Castanopsis. Two regeneration types of woody species in either the early successional (15–50 years), the mid-successional (40–80 years), or the late-successional (80–180 years) stage were classified. Relatively high species diversity was found in the seral phase at the three study sites. The late-successional stage was commonest where human disturbance was least evident. Poor soil chemical properties under pioneer Pinus were seen as a limitation to plant growth, while the abundance of Alnus at the early stage led to an improved level of organic matter and nitrogen.     

蚕豆幼胚珠培养及与近缘种杂交的研究

为提高蚕豆的抗病性、抗虫性和着荚性,人们试图将其与野生近缘种进行种间杂交,但因杂交幼胚早期败育,至今未获成功。解决杂交幼胚的发育问题已成杂交成功的关键。作者以自交授粉15天的幼胚珠为材料,确立了由幼胚珠离体培养经继代直接成苗的方法,获得可正常开花结实的完整植珠。以蚕豆为母本与5个近缘种进行了种间杂交,对杂交20天的杂交幼胚珠进行了离体培养,经愈伤组织再分化形成试管苗。为克服蚕豆与近缘种杂交幼胚的败育进行了探索。     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/<Emphasis Type="Italic">FRT</Emphasis> site-specific recombination system

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.