Dynamics of Proliferation and Differentiation after Transplantation of Hematopoietic Cells in Mouse / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of Listeria monocytogenes infection on hematopoietic stem and progenitor cell (HSPC) composition, cell cycle and cell colony-forming ability in mouse bone marrow.@*METHODS@#The C57BL/6J mice were divided into infected group and control group. The mice in injected group were infected intraperitoneally with 6.7×10 CFU Listeria monocytogenes,while the mice in control group were injecfed with PBS of same volume.The serum levels of IFNγ were detected at different time points. After 24 hours, the HS/PC composition, cell cycle and cell colony-forming ability in bone marrow of mice were measured, and the difference between the control group and the infected group was statistically analyzed.@*RESULTS@#Serum IFNγ levels peaked at 24 hours after infection with Listeria monocytogenes. After 24 h, the proportion of LSK, LSK in S phase, and short-term hematopoietic stem cells (ST-HSC) in the infected group were significantly higher than those in the control group (P＜0.001), long-term hematopoietic stem cells (LT-HSC) and the proportion of LT-HSC in S phase were significantly increased (P＜0.01), and the cell colony-forming ability of bone marrow significantly decreased (P＜0.01). [WTHZ]Conclusion: [WTB1]After infection with Listeria monocytogenes, bone marrow hematopoietic stem cells enter the proliferative state from rest, the cell colony-forming ability decreases, suggesting that Listeria monocytogenes infection can cause hematopoietic stem cell depletion.

Establishment of gene transduction system in geldanamycin producer - Streptomyces hygroscopicus 17997 and its application for gene disruption experiment / 生物工程学报

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.