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An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.

We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium, using the primary murine embryo fibroblast as feeder cells, and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method". We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively. Compared with routine method of establishing ES cell lines, the improved method made distinct differences, increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%, that of C57BL/6J mouse from 3.7% to 13.3%, that of BALB/c mouse from 2.9% to 19.4%. We tested the appropriate dispersing occasion, that is proliferating period of the ICM, affected the formation of ES clones and the ratios of ES cell lines established. It was shown that the most appropriate dispersed occasion for the ICM of 129/ter, C57BL/6J, BALB/c, KM and ICR mice was 4-6 d, 3-3.5 d, 4 d, 4-5 d, 4-5 d after ICM proliferation respectively. At the same time, the effects of the concentration of digestive enzyme buffer were discussed. It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance. It was shown that 'the series dispersed method' was much better than 'the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones. Compared with the routine ES cell culture medium containing mLIF, the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype, but also promoted the attachment and growth of ES cells. This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.

The linearized plasmid pEGFP-N3 was electroporated into three different mouse ES cell lines MESPU-13, MESPU-35 and MESPU-62 derived from 129/ter, C57BL/6J and BALB/c mouse strains respectively. Resistant clones were selected in the presence of G418 and then were identified under the fluorescence microscope through blue exciting light. Positive green clones were primarily expanded and further sorted using FACS(fluorescence activated cell sorter). Finally five EGFP stable integrated cell strains were obtained and were expanded (2 strains from 129/ter, 1 strain from C57BL/6J and 2 strains from BALB/c). Each of the five cell strains presents high proliferation growth rate and typical morphology characters of ES cells and their colonies. More than 85% cells of each cell strain contain normal diploid karyotype. Then some analysis such as the AP (alkaline phosphatase) staining, oct4 gene expression assay, embryonic body formation and differentiated test in vivo and in vitro were made. The results indicated that the stable labeled ES cell strains had the normal karyotypes and maintained the ES cell typical characteristics.

RNA interference is a phenomenon of gene silencing directed by double-stranded RNA. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in Caenorhabditis elegans, Drosophila melanogaster, etc. For mammalian cells, dsRNA directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma (EC) cells. Our previous work proved the existence of RNAi effect for reporter gene GFP and endogenous gene Oct4 in undifferentiated murine ES cells. Yet in other kinds of mammalian cells, because of the existence of interferon pathway, long dsRNA will induce the cells to shutdown global protein translation and go to apoptosis. Therefore, dsRNA longer than 30 bp cannot be used to induce specific gene knockdown effect in these cells. Elbashir et al found that in vitro synthesized small interfering RNA (siRNA) (19-23 nt) could induce potent RNAi as effective as long dsRNA without showing unspecific effect, so that the interferon pathway could be bypassed. It was shown that during RNAi process, long dsRNA was first degraded into 19-23 nt siRNA and then recruited into RISC (RNA induced silencing complex) to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about a week. Recently, it has been shown that U6 promoter directed small hairpin RNA (shRNA) can induce potent gene knockdown effect in murine P19 Embryonic Carcinoma cell. The RNAi effect of U6 promoter-driven shRNA corresponding to Green Fluorescence Protein (GFP) in COS-7 cells was checked. And it was found that the U6 promoter-driven shRNA for GFP can specifically and potently knockdown the GFP's expression in COS-7 cells. The result established the feasibility of using RNAi technique directed by U6 promoter-driven shRNA to study genes' function in COS-7 cell line.

A new method for establishing ES cell lines from 129/ter. C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM) for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.

Several methods and processes of establishment and culture of BALB/c mouse ES cell lines were discussed detailedly. A new method to establish and culture ES cell lines derived from BALB/c mouse was set up successfully using mouse embryonic fibroblast feeder layer and rat-heart-cell-conditioned medium (RH-CM). These culture conditions not only maintain the undifferentiated state and normal diploid karyotypes of BALB/c mouse ES cells effectively, but also maintain a series of their characteristics of murine stem cells. two different kinds of digestive methods and Two kinds of digestive juice with different concentrations were designed to dissociate proliferous inner cell mass (ICM) and ES cell colonies derived from dissociative ICM. two different kinds of digestive methods are "single time dissociation method" and "several times dissociation method", two kinds of digestive juice are 0.25% Trypsin-0.04% EDTA and 0.05% Trypsin-0.008% EDTA. At the same time, appropriate dissociated occasion of ICM and the effect of RH-CM on establishment and culture of BALB/c mouse ES cell lines were discussed. The results suggested that it is a reasonable method to establish BALB/c mouse ES cell lines using low concentration digestive juice and "several times dissociation method" to dissociate ICM after 4 days' proliferation. Judged by the form of ES cells and its colonies, proliferous capability, karyotypes examine, alkaline phosphatase activity assay and differentiation capability in vitro and in vivo, the 9 ES cell lines that we established satisfied the all traits of murine ES cell line.

17 kinds of conditioned media were selected by plating tests, propagation and other tests with two cell lines, C-19-2 and MESPU-13. The results suggested that the rat heart cells conditioned media (RH-CM) can inhibit the spontaneous differentiation effectively, maintain the normal diploid karyotypes and promote adherence and proliferation of mouse ES cells. The ES cells were propagated in RH-CM to the 20th passage remaining their pluripotent in vivo and in vitro differentiation ability. RH-CM can be used as supplement of ES cell media. ES cells which are cultured in media containing 70% RH-CM and on PMEF feeder can maintain their undifferentiation state and diploid karyotype. RT-PCR detection suggested that there was mLIF expression in rat heart cells.