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this journal aims to bring together innovative new research across the fields of cell and molecular biology, immunology, cancer, clinical medicine, and pathophysiology to help researchers find the answers they need to get ahead in their careers. we welcome paper submissions that are original, innovative and address significant topics in the fields of cancer, immunology and cancer therapy. we also encourage the inclusion of discussion of the latest research in the fields of cell and molecular biology, immunology, cancer, clinical medicine and pathophysiology.
in this study, we evaluated the ability of different viral ires elements to initiate protein synthesis in various eukaryotic cell-free systems (wheat germ, sf21, cho and k562 cells). the efficiency of iress from the encephalomyocarditis virus (emcv), rhopalosiphum padi virus (rhpv) and cricket paralysis virus (crpv) genomes were investigated. the main goal of the study was to identify iress that are universally applicable in a range of eukaryotic cell-free protein expression systems. from the iress tested, the igr ires from the crpv genome was the most efficient across all cell-free systems.
the expression of the crispr/cas9 system is under tight control of the spcas9-grna complex, and is activated by the host transcription factor sigmae, allowing efficient expression in a wide range of hosts including escherichia coli, bacillus subtilis, and even human cells. expression of the crispr/cas9 in a host cell is often required for the cell to become sensitive to the action of the cas9 protein. therefore, it is very important that the cells that express the cas9 protein are compatible with the host cell type. in the experiments described here, we created a spcas9 protein for use in the model organism bacillus subtilis. we cloned the spcas9 gene into a vector that allows homologous recombination in b. subtilis. we then introduced the resulting plasmid into b. subtilis, and obtained a stable cell line that expressed a spcas9-grna complex under the control of a sigmae promoter. by introducing a grna that targets the staphylococcal enterotoxin b gene, we created a cell line that can be used to silence any gene in the genome. we also constructed a cas9 plasmid that expresses the sgrna transcript that encodes the grna under control of the sigmae promoter, and a plasmid that expresses the crrna under control of the arabad promoter. the expression levels of the cas9 protein and the sgrna are much higher in the cell line containing the spcas9 plasmid than in the cells containing the sigmae-cas9 plasmid. this difference allows us to perform more efficient genome editing in the new cell line. the cell line containing the spcas9-grna plasmid can also be used to express the cas9 protein and the grna in human cells.. why is there no thermotolerance in bacillus subtilis? invertase gene expression in bacillus subtilis under sigmae-control an. m. lambregts; e. van vliet; h. weening. molecular microbiology. 2007;57(7):1475-1488. the crpv igr ires bypasses the first 60 nucleotides of the 5’untranslated region (5’utr), making it an excellent candidate to insert a functional gene in the 5’utr of a viral or a cellular mrna. in this chapter we describe the design and cloning of a plasmid that expresses a functional beta-lactamase gene under the control of the crpv igr ires in the 5’utr of a chloramphenicol-resistance gene. we also describe the in vitro expression and translation of the beta-lactamase using a cell-free translation system and cell extracts from human cells and a variety of insect and mammalian cell lines. t. subramaniam; s. subramaniam; b.h. chan; d. h.g. mcmillan; l.e. sanchez; l. p. lee; e.j. fitzgerald; a. maio; d. santagati; g. barbero. proc. natl. acad. sci. u.s.a. 2007;104(46):18522-18527. a. r. austin; d. kirk; a. c. tarn; t. wong; a. w. cook; m. s. lomonossoff; s. l. bradlow; p. bradbury; m.
dr. christine fischetti is a senior consultant in the department of medical microbiology, clinical immunology, and medical genetics at the university of washington school of medicine. she obtained her phd in the department of microbiology and immunology at the university of washington. her research focuses on the use of human-derived tumor cell lines, both as models and as platforms for drug discovery. in addition, she uses these cells as tools for the investigation of the key features of tumor biology that include the induction and maintenance of an immune suppressive tumor microenvironment. she has authored or co-authored over 85 peer-reviewed publications.
the authors would like to thank annie allaert and catherine fontaine of microscopic image department of igbmc for their assistance with the immunohistochemistry experiments and prof. j. claude chowen of the department of microbiology at the university of washington for his valuable advice with the viability of the cell-free system.
de novo synthesized active luc was monitored after 3 h of incubation at 27c (sf21 cell extract) and 33c (cho and k562 cell extracts), respectively. cell-free protein synthesis was performed using the optimized vector containing the crpv igr ires harboring an aug-to-gcu mutation of the initiation codon in the easyxpress pix3.0 vector backbone. protein yields were normalized to the reaction with the highest yield of active luc (=100%) for each cell-free system. yields of active luc were determined from three independent experiments using a luc reporter assay and the corresponding standard deviations were calculated.