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Expression Cloning (expression + cloning)
Selected AbstractsMammalian Expression Cloning of Two Human Trophoblast Suppressors of Major Histocompatibility Complex GenesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2001J. A. PEYMAN PROBLEM: Human trophoblasts suppress interferon-, (IFN-,)-simulated expression of major histocompatibility complex (MHC) class II genes and thereby protect the conceptus from maternal immune attack. The mechanism of this suppression is poorly understood. METHOD OF STUDY: IFN-,-responsive HeLa cells were stably transfected with trophoblast cDNA expression libraries and screened by negative immunoselection with an antibody to HLA-DR. RESULTS: Two suppressor cDNAs were isolated. One encoded the untranslated RNA trophoblast STAT utron (TSU), which blocked STAT1 nuclear translocation and can theoretically form triplex RNA,DNA at the class II transactivator gene promoters. The other encoded the N-terminal 28 residues of chorionic somatomammotropin (hCS). TSU-related genes were detected in human and macaque, but not in mouse, genomic DNA. CONCLUSIONS: The genetics of two human trophoblast MHC suppressors suggest that these functions have been gained in human placenta in recent evolutionary history. TSU and hCS play critical roles in suppression of MHC genes, which may lead to silencing by DNA methylation. [source] Isolation of flagellated bacteria implicated in Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 10 2007L. Wayne Duck BS Abstract Background: Serologic expression cloning has identified flagellins of the intestinal microbiota as immunodominant antigens in experimental colitis in mice and in individuals with Crohn's disease (CD). The present study was done to identify the microbial source of such flagellins. Methods: Using a variety of isolation and culture approaches, a number of previously unknown flagellated bacteria were isolated. Based on 16S ribosomal DNA sequences, these bacteria fall into the family Lachnospiraceae of the phylum Firmicutes. Results: Serum IgG from patients with CD and from mice with colitis reacted to the flagellins of these bacteria, and only their flagellins, whereas serum IgG from controls did not. The sequence of these flagellins demonstrate conserved amino- and carboxy-terminal domains that cluster phylogenetically and have a predicted 3D structure similar to Salmonella fliC, including an intact TLR5 binding site. The flagellin of 1 of these bacteria was likely O -glycosylated. Conclusions: The conserved immune response in both mouse and human to these previously unknown flagellins of the microbiota indicate that they play an important role in host,microbe interactions in the intestine. (Inflamm Bowel Dis 2007) [source] Identification of antigenic targets of paraproteins by expression cloning does not support a causal role of chronic antigenic stimulation in the pathogenesis of multiple myeloma and MGUSINTERNATIONAL JOURNAL OF CANCER, Issue 2 2007Klaus-Dieter Preuss Abstract Antigenic targets of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) paraproteins have been suggested to play an important role as growth stimulators in the pathogenesis of these neoplasms. To identify such targets, we screened cDNA libraries from human testis, lung and breast cancer, bovine and porcine muscle and wheat germ for reactivity with paraproteins in the sera from 115 patients with MGUS and MM. Of >6 × 108 paraprotein,antigen interactions screened, an IgA paraprotein from a female patient bound to sperm-specific cylicin-2, and 3 IgG paraproteins bound to tripeptidyl-peptidase-II (TPP-2), insulin-like growth-factor binding-protein-2 (IGFBP-2) and porcine kinesin. Specificity was confirmed by reverse Western blots using recombinant antigens. The broad spectrum of auto-, allo- and heteroantigens as targets of human paraproteins in patients without signs of chronic antigenic stimulation renders a causal role of the antigenic stimulus in the pathogenesis of MGUS and MM unlikely. © 2007 Wiley-Liss, Inc. [source] Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 6 2004Kazue Nakashima Abstract We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti-TROP2 antibodies (s-TROP2-Abs) in patients with esophageal SCC, the presence of s-TROP2-Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s-TROP2-Abs. Positivity in terms of s-TROP2-Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s-TROP2-Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers. © 2004 Wiley-Liss, Inc. [source] Enterocytin: A new specific enterocyte marker bearing a B30.2-like domainJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Stéphane Parnis Enterocyte differentiation is correlated to the expression of specific proteins which only a few of them are identified. In this study, we characterize a new marker of enterocyte differentiation using monoclonal antibodies. We showed that small intestinal enterocytes specifically express a new 47 kDa protein named Enterocytin. Expression of this protein increase along the crypt-villus axis and it is concentrated in the terminal web, lateral plasma membrane domain, and nucleus membrane of mature enterocytes. A 1.8-kb cDNA of Enterocytin was isolated by expression cloning from a cDNA library of rabbit small intestine. The amino acid sequence obtained shows an N-terminal region with a coiled-coil structure and a B30.2-like domain in the C-terminus region. By co-transfection and immunoprecipitation procedures on Cos cells, it was observed that the coiled-coil domain is involved in the homodimerization of Enterocytin. In the human intestine, a similar 47 kDa protein was detected, exclusively in the small intestinal enterocytes. In addition, expression of this protein in Caco2 cells is correlated with the state of differentiation of these cells. The restricted expression of Enterocytin in the intestine and its localization in mature cells suggest that it may contribute to the differentiation processes and maintenance of the enterocytic polarity. J. Cell. Physiol. 198: 441,451, 2004© 2003 Wiley-Liss, Inc. [source] Screening of chondrogenic factors with a real-time fluorescence-monitoring cell line ATDC5-C2ER: Identification of sorting nexin 19 as a novel factorARTHRITIS & RHEUMATISM, Issue 11 2009Akinori Kan Objective To establish a cell culture system for noninvasive and real-time monitoring of chondrogenic differentiation in order to screen for chondrogenic factors. Methods The optimum reporter construct transfected into chondrogenic ATDC5 cells was selected by a luciferase reporter assay and fluorescence analysis during cultures with insulin. The established cell line was validated according to its fluorescence following stimulation with SOX proteins, bone morphogenetic protein 2 (BMP-2), or transforming growth factor , (TGF,) and was compared with the level of messenger RNA for COL2A1 as well as with the degree of Alcian blue staining. Screening of chondrogenic factors was performed by expression cloning using a retroviral expression library prepared from human tracheal cartilage. The expression pattern of the identified molecule was examined by in situ hybridization and immunohistochemistry. Functional analysis was performed by transfection of the identified gene, the small interfering RNA, and the mutated gene. Results We established an ATDC5 cell line with 4 repeats of a highly conserved enhancer ligated to a COL2A1 basal promoter and the DsRed2 reporter (ATDC5-C2ER). Fluorescence was induced under the stimulations with SOX proteins, BMP-2, or TGF,, showing good correspondence to the chondrogenic markers. Screening using the ATDC5-C2ER system identified several chondrogenic factors, including sorting nexin 19 (SNX19). SNX19 was expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression. The gain-of-function and loss-of-function analyses revealed a potent chondrogenic activity of SNX19. Conclusion We established the ATDC5-C2ER system for efficient monitoring of chondrogenic differentiation by fluorescence analysis, and we identified a novel chondrogenic factor (SNX19) using this system. This system will be useful for elucidating the molecular network of chondrogenic differentiation. [source] SEREX identification of new tumour-associated antigens in cutaneous T-cell lymphomaBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2004T.B. Hartmann Summary Background Cutaneous T-cell lymphoma (CTCL) is a clonal lymphoproliferative disorder of mainly CD4+ T cells, with primary manifestation in the skin. Objectives To detect new CTCL-associated antigens for immunological therapies and to define their specificity in terms of RNA expression and seroreactivity. Methods A newly constructed CTCL cDNA phage library was screened and cross-reactivities against the detected clones were tested using 15 mycosis fungoides and six Sézary syndrome sera. The mRNA expression of the identified genes was analysed by reverse transcription,polymerase chain reaction (RT,PCR) using 22 tumour tissues, nine cell lines and up to 29 different types of normal tissue. Results We identified nine different tumour antigens (HD-CL-01 to HD-CL-09) of which seven clones had high homology to genes with known functions. Several of these genes had previously been associated with cancer, namely inositol 1,4,5-triphosphate 5-phosphatase, vimentin, aldose reductase and elongation factor-1,. Variations in the deduced protein sequences were observed in three cases, mostly due to variations in protein length. The individual clones were recognized by up to 56% of patients' sera, while control sera were negative except in one case. Using RT,PCR, we found a frequent expression of these new tumour antigens in tumour specimens (26,100%). In contrast to humoral specificity, specific mRNA was also detected in selected normal tissues (29,89%). Conclusions SEREX (serological identification of antigens by recombinant expression cloning) identified multiple tumour-associated antigens in CTCL. The serological specificity and the high percentage of reactive sera of CTCL patients against several clones suggest these genes as potential targets for diagnostic and prognostic purposes. [source] | ||||||||||