Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

High expression of human serum albumin in milk of transgenic mice directed by the goat β-casein gene promoter region

We have constructed a mammary gland expression vector that contained the goat β-casein gene promoter, 5′upstream regulatory region, exons 1,2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injection of the vector into mouse tail veins showed that the recombinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F₀ mice were obtained. Of the 33, 8 mice (5♀, 3 ♂) were transgenic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, respectively.     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

Two-dimensional crystallization and preliminary electron crystallographic result of partially purified F<Subscript>0</Subscript> from porcine mitochondria

After removal of cytoplasmic sector F₁ from submitochondrial particles of F₀F₁-ATP synthase complex with guanidine hydrochloride, the transmembrane sector F₀ was specifically extracted from the stripped membranes in the presence of detergent CHAPS and partially purified. Two-dimensional crystals were produced by the reconstitution of the partially purified F₀ into asolectin and microdialysis. The obtained crystals are able to diffract to 2 nm. The projection map of the negatively stained crystal shows that the crystal has p42₁2 symmetry, lattice constant, a=b=14.4 nm. A unit cell contains four F₀ molecules.     

50例中国人IX因子基因的限制性片段长度多态性

用IX因子基因内探针F9(VⅢ)对TaqI,BamHI和EcoRI酶切的50例中国人基因组DNA进行杂交分析。结果表明,所有个体经TaqI酶切的杂交片段为4.5kb和1.8kb,BamHI和EcoRI酶切的杂交片段分别为23kb和5.0 kb。基因组DNA样本中未发现限制性片段长度多态性(RFLP),这与欧美国家的民族群体中存在着IX因子基因内TaqI和BamHI的RFLP的结论不同。造成不同种族间DNA水平差异的原因,很可能与长期在不同地理环境中的进化适应有关。     

Identification of RAPD marker linked with fertility-restoring gene of cytoplasmic male sterile lines in upland cotton

Bulked segregant analysis was employed to construct two mixed DNA pools to screen the RAPd marker linked with the fertility-restoring gene(Rf _i) of upland cotton. A total of 425 arbitrary 10-mer oligonucleotide primers were screened on two DNA pools, bulked male fertile and sterile DNAs isolated from BC₃ segregating population of (0-613-2R X Simian No. 3). Three primers produced repeatable polymorphisms between the paired bulks and their parents. DNA was extracted and amplified with these three primers for 92 plants of (Zhong 12A-1 × 0-613-2R)F₂. Based on the male fertility scoring and RAPD amplification, it is found that one RAPD marker fragment designated OPV-15₃₀₀ was linked with the fertility-restoring gene (Rf₁) with a recombination value of 13.0±2.57%.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

Direct observation of the clockwise light-driven rotation of F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase complex

The purified thermophilic bacterium PS3 F₁ β _10×His-tag is inserted into the F_oF₁-ATP synthases of chro-matophores isolated from photosynthetic bacteria Rhodo-spirillum rubrum. The studies of biochemical properties of the hybrid chromatophores show that they have both protons-driving capability and photophosphorylation. The fluorescent actin filaments, as a marker of its orientation by video-microscopic experiment, are connected via Maleimido-C₃-NTA to the reconstituted β_10×His-tag of F_oF₁-ATP synthases. The clockwise rotation of F_oF₁-ATP synthases driven by light is observed directly when viewed from the F_o side to F₁. This system should be valuable for further studying the coupling property of F_oF₁-ATP synthase.     

Building of hFⅨ transgenic mice by spermatogenic cells

Human FⅨ expression vector pCMVⅨ was packaged by effectene^TM reagent and injected into mice seminiferous tubules with glass pipettes.The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies.There were 2(4%) mice being integrated with hFⅨ gene into chromosomes.4.6ng/mL of hFⅨ protein was expressed in plasma of one mouse,which was tested by ELISA.We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method.Meanwhile,it has also been proved to be an alternative choice for mammary gland bioreactor.     

The planimetric unfold method of fullerene cage structure

Two kinds of planimetric diagrams, which consist of the boat form F₆ and F₅, the storm petrel form F₆ and F₅, respectively, were proposed to express the geometric structure of fullerene cage in this study. There are two chief advantages using the diagrams: (ⅰ) the spatial symmetrical characteristic of fullerene cage is not destroyed; (ⅱ) the coordination forms of F₅ and F₆in the structure can be clearly expressed. This work has laid the foundation for studying the structural geometry of fullerene cage and its quantum chemistry and property.     

Tagging major quantitative trait loci for sheath blight resistance in a rice variety, Jasmine 85

This study was conducted with a clonal F₂ population of rice from a cross between Jasmine 85, a resistant variety, and Lemont, a susceptible cultivar. The rice plants belonging to each F₂ clone were divided into two plots, which were put in two replicates, respectively. Clonal parents were tested as controls. The plants were inoculated by short toothpicks incubated with RH-9, a virulent isolate of the pathogenic fungus,Rhizoctonia solani, which causes rice sheath blight. The extreme resistant and susceptible clonal lines were selected for construction of resistant and susceptible DNA pools, respectively. A total of 94 polymorphic markers evenly distributed on 12 rice chromosomes were used for bulked segregant analysis, three positive ones were found polymorphic between the two DNA pools, and three major QTLs for sheath blight resistance, Rh-2, Rh-3 and Rh-7, were identified. The three major QTLs were located on chromosomes 2, 3 and 7, and could explain 14.4%, 26.1% and 22.2% of the phenotypic variation.     

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.     

Comparative analysis of A, B, C and D genomes in the genus Oryza with Cot-1 DNA of C genome

Fluorescence in situ hybridization （FISH） was applied to somatic chromosomes preparations of Oryza officinalis Wall. （CC）, O. sativa L. （AA）xO. officinalis F1 hybrid （AC）, backcross progenies BC1 （AAC and ACC）, O. latifolia Desv. （CCDD）, O. alta Swallen （CCDD） and O. punctata Kotschy （BBCC） with a labelled probe of Cot-1 DNA from O. officinalis. In O. officinalis, the homologous chromosomes showed similar signal bands probed by Cot-1 DNA and karyotype analysis was conducted based on the band patterns. Using no blocking DNA, the probe identified the chromosomes of C genome clearly, but detected few signals on chromosomes of A genome in the F1 hybrid and two backcross progenies of BC1. It is obvious that the highly and moderately repetitive DNA sequences were considerably different between C and A genomes. The chromosomes of C genome were also discriminated from the chromosomes of Dand B-genome in the tetraploid species O. latifolia, O. alta and O. punctata by Cot-1 DNA-FISH. Comparison of the fluorescence intensity on the chromosomes of B, C and D genomes in O. latifolia, O. alta, and O. punctata indicated that the differentiations between C and D genomes are less than that between C and B genomes. The relationship between C and D genomes in O. alta is closer than that of C and D genomes in O. latifolia. This would be one of the causes for the fact that both the genomes are of the same karyotype （CCDD） but belong to different species. The above results showed that the Cot-1 DNA had a high specificity of genome and species. In this paper, the origin of allotetraploid in genus Oryza is also discussed.     

The concentration quenching characteristics of 5D3-7FJ and 5D4-7FJ (J=0―6) transitions of Tb3+ in YBO3:Tb3+ phosphor

The photoluminescence quenching behaviors of ^5D3-^7Fj and ^5D4-^7Fj （J = 0—6） transitions of Tb^3＋ in YBO3：Tb under 130—290 nm excitation were systematically investigated. The results revealed that the quenching concentrations of both ^5D3-^7Fj and ^5D4-^7Fj transitions of Tb^3＋ in YBO3：Tb were mainly dependent on excitation wavelength. Particularly, the quenching concentrations of ^5D4-^7Fj transitions of Tb^3＋ under 130—290 nm excitation were correlated with excitation bands of YBO3：Tb. The quenching concentrations of ^5D3-^7Fj transitions remained at low concentration （2%） under 186—290 nm excitation and then increased gradually with energy of incoming excitation photon when excited at 130—186 nm. This dependence should be involved in their excitation mechanisms and quenching pathway in particular excitation region.[第一段]     

小球藻(Chlorella vulgaris)抗铵品系的紫外诱变选育

【目的】获得耐受高铵浓度的优良养殖小球藻(Chlorella vulgaris)品系,可以有效防止其在养殖过程中被轮虫,纤毛虫等污染生物吞食。【方法】首先对小球藻进行紫外线诱变,然后用高浓度NH_4HCO_3作为筛选压力对其进行筛选。【结果】筛选到具有生长优势的小球藻突变株B4和C6,培养12d,在NH_4HCO_3浓度为800mg/L、1 000mg/L、1 200mg/L条件下,B4和C6的生物量比出发藻株分别提高41.18%和9.54%,32.64%和29.41%,37.74%和22.85%;对比出发藻株,B4和C6的最大光能转化效率有显著提高。【结论】突变株B4和C6在高铵培养条件下更能显示生长优势,且它们的生长优势可能来自光合效率的提高。     

Construction of AFLP-based genetic linkage maps for the Chinese shrimp Fenneropaeneus chinensis

Fenneropaeneus chinensis is an important species in marine fishery resources and aquaculture in China. A genetic linkage map is essential for improving the efficiency of its breeding by marker-assisted selection and identifying commercially important genes. Linkage maps of F. chinensis were constructed with an F2 mapping population （110 progenies） using amplified fragment length polymorphic （AFLP） marker in this study. Fifty-five AFLP primer combinations produced 532 AFLP markers fitting for map strategy in mapping family. The markers with 3：1 segregating ratios were analyzed using F2 intercross model for the common linkage map, while the markers with 1：1 ratio were analyzed using the pseudo-testcross strategy. The maps of male, female and common were constructed. The female map included 103 markers that formed 28 linkage groups, covering a total length of 1090 cM. All markers were linked with the linkage groups. Segregation distortion was observed for 6 of 103 markers in the female map. The average distance between markers was 14.53 cM and ranged from 4.4 to 24.8 cM. The male map included 144 markers that formed 35 linkage groups. Ten markers remained unlinked in male map. Segregation distortion was observed for 7 of 144 markers in the male map. The total distance of male map covered 1617 cM. The average distance between markers was 16.36 cM. The male map was 32.6% longer than the female map, which may reflect sex-specific recombination rates in Chinese shrimp. The common map was composed of 216 markers, including in 44 linkage groups covering a total distance of 1772.1 cM. Two markers remained unlinked. No distorted markers of 216 markers were shown in the common map. The distance between markers was 10.42 cM. An average estimated genome size for the Chinese shrimp was 2420 cM, which was consistent with the relative size of the Penaeid genome. The distribution of AFLP markers was relatively even in chromosomes of Chinese shrimp maps. The linkage analysis presented in this work provided some insight     

Identification of an AFLP marker linked to the stripe rust resistance geneYr10 in wheat

AFLP analysis of near-isogenic lines of the stripe rust resistance gene Yr10 was carried out with 6 PstⅠ- primers and 10 TaqⅠ-primers with the donor parent of Yr10 gene as the check. A total of about 4200 distinguishable bands were amplified, of which 5 were stable. The genetic linkage of the 5 polymorphic DNA fragments with the target gene were tested preliminarily on a segregating F₂ population derived from a cross between the gene donor parent “Moro” and susceptible cultivar “Mingxian 169”. The DNA fragment PT0502 was found closely linked to the Yr10 gene and cloned and sequenced. Based on the sequence specific primers for PCR were designed and synthesized. Genetic linkage analysis with 195 segregating F₂ plants indicated that the genetic distance was 0.5 cM between the main product SC₂₀₀ fragment produced by PCR with the primers and the Yr10 gene. The primers can be used to detect the Yr10 gene quickly, effectively and exactly.     

双抗体夹心ELISA法测定人凝血因子IX蛋白

用双抗体夹心ELISA法测定人凝血因子IX蛋白。在转入了外源人凝血因子IX基因的CHO细胞和HSF细胞的培养液中均测得一定量的IX因子蛋白,其量相当于正常人血浆IX因子蛋白的0.89%～3.81%,而对照细胞均为阴性结果。对一例血友病B患者(IX:C<0.1%)的分析结果表明,其血浆中含有的IX因子蛋白量相当于正常人的52.3%,因而将此病例归属于血友病B~R型。本法灵敏度高、特异性强、重复性好,可与一期法相互补充,应用于临床血友病B的诊断、分类、家系分析及携带者的检出。     

Expression of active human clotting factor IX from recombinant DNA clones in mammalian cells

Haemophilia B, or Christmas disease, is an inherited X-chromosome-linked bleeding disorder caused by a defect in clotting factor IX and occurs in about 1 in 30,000 males in the United Kingdom. Injection of factor IX concentrate obtained from blood donors allows most patients to be successfully managed. However, because of impurities in the factor IX concentrate presently in use, this treatment involves some risk of infection by blood-borne viruses such as non-A, non-B hepatitis and the virus causing acquired immune deficiency syndrome (AIDS). Because of the recent concern about the increasing incidence of AIDS amongst haemophiliacs, a factor IX preparation derived from a source other than blood is desirable. Here, we report that after introduction of human factor IX DNA clones into a rat hepatoma cell line using recombinant DNA methods, we were able to isolate small amounts of biologically active human factor IX.