Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.     

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.     

Sludge Reduction with a Novel Combined Worm-reactor

In China, as a result of economic, environmental and regulation factors, excess sludge treatment and disposal represents a rising challenge for small and mid-scale municipal wastewater treatment plants (WWTPs). Although the presence of worms in aerobic wastewater treatment may lead to substantial sludge reduction, the practical application is still uncontrollable because of unstable worm growth. To overcome unstable worm growth in activated sludge process, a combined worm-reactor, consisting of sections for both free-swimming and sessile worms, was developed to enhance worm growth. This plug flow type of worm-reactor has the sessile worm growing section filled with plastic carriers, and recirculation of sludge from the section for sludge settling was carried out in order to avoid worm washing out. Sludge reduction of treating the discharged excess sludge using this worm-reactor was investigated during 53 days. In the start-up phase the combined worm-reactor was inoculated with Tubificidae. Tubificidae mainly occurred in the carriers and on the bottom of the worm-reactor, however, neither Aeolosoma nor Nais was found in the worm-reactor. Results clearly showed that the average sludge reduction was 48%, and the COD removal was low, about 8%. High NH₄⁺ -N or NO₂⁻ -N concentrations or their combination may limit the growth of Tubificidae. Few PO ₄³⁻-P release into the effluent was observed in the worm-reactor. However, results obtained from this study cannot unequivocally be attributed to the presence of worms because it did not include a control system, and thus much more work will be needed in this concept in the further research.     

New concept of contaminant removal from swine wastewater by a biological treatment process

Pollution from concentrated animal feeding operations (CAFOs) are the most serious pollution source in China now, and swine wastewater contains high concentrations of nutrients such as chemical oxygen demand (COD), biochemical oxygen demand 5 (BOD₅), ammonium, and emergent contaminants related to public health. Biological processes are the most popular treatment methods for COD and ammonium removal. Considering the low operation cost, easy maintenance and high removal rate of contaminants in recent years, nitrogen removal via nitrite and real-time control processes using oxidation-reduction potential (ORP) and/or pH as parameters to control the aerobic and anaerobic cycles of a system has received much attention for animal wastewater treatment. During the biological treatment process, the emergent contaminants such as estrogen, antibiotics, and disinfection reagents have been the focus of research recently, and degradation bacteria and resistance bacteria have also been extracted from activated sludge. The microbial analysis technique is also advancement in the field of biodegradation bacteria and resistance bacteria. All of these advancements in research serve to improve wastewater treatment and decrease environmental hazards, especially for using manure as a fertilizer source for crop production.     

New concept of contaminant removal from swine wastewater by a biological treatment process

Pollution from concentrated animal feeding operations (CAFOs) are the most serious pollution source in China now, and swine wastewater contains high concentrations of nutrients such as chemical oxygen demand (COD), biochemical oxygen demand 5 (BOD5), ammonium, and emergent contaminants related to public health. Biological processes are the most popular treatment methods for COD and ammonium removal. Considering the low operation cost, easy maintenance and high removal rate of contaminants in recent years, nitrogen removal via nitrite and real-time control processes using oxidation-reduction potential (ORP) and/or pH as parameters to control the aerobic and anaerobic cycles of a system has received much attention for animal wastewater treatment. During the biological treatment process, the emergent contaminants such as estrogen, antibiotics, and disinfec-tion reagents have been the focus of research recently, and degradation bacteria and resistance bacteria have also been extracted from activated sludge. The microbial analysis technique is also advancement in the field of biodegrada-tion bacteria and resistance bacteria. All of these advance-ments in research serve to improve wastewater treatment and decrease environmental hazards, especially for using manure as a fertilizer source for crop production.     

Transgenic silkworms (<Emphasis Type="Italic">Bombyx mori</Emphasis>) produce recombinant spider dragline silk in cocoons

Spider dragline silk is a unique fibrous protein with a combination of tensile strength and elasticity, but the isolation of large amounts of silk from spiders is not feasible. In this study, we generated germline-transgenic silkworms (Bombyx mori) that spun cocoons containing recombinant spider silk. A piggyBac-based transformation vector was constructed that carried spider dragline silk (MaSp1) cDNA driven by the sericin 1 promoter. Silkworm eggs were injected with the vector, producing transgenic silkworms displaying DsRed fluorescence in their eyes. Genotyping analysis confirmed the integration of the MaSp1 gene into the genome of the transgenic silkworms, and silk protein analysis revealed its expression and secretion in the cocoon. Compared with wild-type silk, the recombinant silk displayed a higher tensile strength and elasticity. The results indicate the potential for producing recombinant spider silk in transgenic B. mori.     

Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos

Incorporation of [3H]thymidine at different concentrations into mouse embryos at early developmental stages was determined by autoradiography. Methods to synchronise the G1-phase of mouse 2- and 4-cell embryos were also investigated. The results showed that the ability of embryos to incorporate [3H]thymidine increased with development. Embryos at the 4-cell stage were not labelled when the concentration of [3H]thymidine was lower than 5 microCi/ml, whereas the nuclei of embryos at morula and blastocyst stages began to show silver grains at a concentration of 0.1 microCi/ml of [3H]thymidine. After 2- and 4-cell mouse embryos were synchronised at the onset of G1-phase by treatment with low temperature or nocodazole, and DNA synthesis was detected by autoradiography, the duration of G1-phase was estimated. The result showed that 43% of the 2-cell embryos had a G1-phase of < or = 1 h, 22% had a G1-phase of < or = 2 h, 22% had a G1-phase of < or = 3 h and 13% had a G1-phase of < or = 4 h. The G1-phase in 85% of the 4-cell embryos was < or = 3 h, that in 8% of embryos was < or = 4 h and that in 7% of embryos was < or = 5 h. The toxicity of nocodazole on mouse embryo development was assessed based on both blastocyst formation and the number of blastomeres, and the results indicated that the effect of nocodazole on embryo development and cell cycle block was dose-dependent. The minimum concentration of nocodazole for metaphase block of mouse late 2-cell embryos was 0.05 microM, and the appropriate concentrations which did not impair development were 0.05-0.5 microM.     

水稻和玉米苗上饲养的稻纵卷叶螟对温度的反应

比较了水稻和玉米苗上饲养的稻纵卷叶螟在不同温度下的存活、生长发育与繁殖情况。结果表明,在24℃和27℃下,稻纵卷叶螟在玉米苗上的发育速率要比在水稻上的慢1—3 d;33℃不会抑制水稻上饲养幼虫的生长发育,但玉米苗上饲养的幼虫在30℃时就受到了抑制,且在33℃下不能发育到成虫。水稻上饲养时,稻纵卷叶螟各阶段的存活率在27—33℃间无显著差异,且36℃时死亡率很高,只有部分幼虫可发育到预蛹阶段;但是玉米苗上饲养时,稻纵卷叶螟在33℃下的存活率显著低于21—30℃,36℃下幼虫不能发育到2龄。水稻和玉米苗上饲养的稻纵卷叶螟的产卵量均表现为在24℃和27℃条件下最高;在30℃和33℃条件下,水稻上饲养出的成虫能产少量卵,而玉米苗上饲养出的成虫却不能产卵。水稻和玉米苗上饲养的稻纵卷叶螟卵的孵化率均在27℃下最高,且两寄主间无显著差异;玉米苗上饲养所得的卵在33℃和36℃下均不能孵化,而水稻上的则少量能孵化。水稻上饲养的稻纵卷叶螟相对要比玉米苗上饲养的耐高温。在24—27℃下用玉米苗饲养的稻纵卷叶螟除历期有所延长外,其它生物学特性与用水稻饲养的无显著差异。