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  • 1
    In: Biology of Reproduction, 2000, Vol.63(5), p.1555-1561
    Description: Abstract We developed a novel promoter-based selection strategy that could be used to produce cell lines representing sequential stages of spermatogenesis. The method is based on immortalization and subsequent targeted selection by using differentiation-specific promoter regions. As an example for this approach, a new murine germ cell line (GC-4spc) was established using a vector construct that contains the SV40 large T antigen and the neomycin phosphotransferase II gene under the control of the SV40 early promoter and a spermatocyte-specific promoter for human phosphoglycerate kinase 2, respectively. The GC-4spc was characterized as a cell line at the stage between preleptotene and early pachytene spermatocytes. Transcription of three germ cell-specific expressed genes, Pgk2, proacrosin, and the A-myb proto-oncogene, were detected in the GC-4spc cell line using reverse transcription-polymerase chain reaction. Furthermore, TSPY (human testis-specific protein, Y-encoded) and PGK2 (human phosphoglycerate kinase 2) promoter regions showed different transcriptional activities in the GC-4spc cell line compared with the spermatogonia-derived cell line GC-1spg. Thus, our strategy could be used for immortalization of cells at specific stages of differentiation, allowing production of a series of cultured cell lines representing sequential stages of differentiation in given cell lineages.
    Keywords: Spermatogenesis ; Testes
    ISSN: 0006-3363
    E-ISSN: 15297268
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  • 2
    Language: English
    In: International Journal of Oncology, March 2001, Vol.18(3), pp.567-574
    Description: In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin α6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, α-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.
    Keywords: Biomarkers, Tumor -- Genetics ; Neoplasm Proteins -- Genetics ; RNA, Messenger -- Metabolism ; Spermatogonia -- Metabolism;
    ISSN: 1019-6439
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  • 3
    Language: English
    In: Biochemical and Biophysical Research Communications, 16 June 2000, Vol.272(3), pp.951-952
    Description: In the article by Hiramoto et al. (1997) (Stage-specific expression of a mouse homologue of the porcine 135 kDa -d-mannosidase (MAN2B2) in type A spermatogonia, Biochem. Biophys. Res. Commun. 241, 439–445) a new homologue to the porcine epididymis-specific 135 kDa -d-mannosidase (MAN2B2) was cloned from a mouse testis cDNA library. Northern blot analysis with RNA of different tissues showed a testis-specific expression by using a mouse MAN2B2 oligonucleotide. In situ hybridization revealed that the mouse MAN2B2 transcript is localized exclusively in spermatogonia. In consequence to this it was proposed by Hiramoto et al. that the mouse MAN2B2 homologue could serve as a marker for spermatogonia. By repeating the published experiments we observed a different expression pattern of the mouse MAN2B2 gene. Northern blot analysis with either testicular RNA from prepubertal males or with testicular RNA from the W/Wv-mutant mouse which lacks germ cells showed an expression of the MAN2B2 gene. Furthermore, Northern blot analysis with RNA from different somatic tissues revealed that the gene is ubiquitously expressed. Therefore, our refinement clearly demonstrates that the MAN2B2 mouse homologue is not specifically expressed in spermatogonia. Copyright 2000 Academic Press.
    Keywords: Biology ; Chemistry ; Anatomy & Physiology
    ISSN: 0006-291X
    E-ISSN: 1090-2104
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  • 4
    Language: German
    Description: Göttingen, Univ., Diss., 2001.
    Source: Networked Digital Library of Theses and Dissertations
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  • 5
    In: Molecular Therapy, 2004, Vol.9(5), p.637
    Description: We have developed a gene switch based on the human transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) and its activation by rosiglitazone. However, ectopic expression of PPARgamma has been demonstrated to convert myogenic cells into adipocyte-like cells and, more generally, may interfere with the physiology of the target tissue. Consequently we modified the DNA-binding specificity of PPARgamma, resulting in a transcription factor that we named PPAR*. We demonstrated by histological and molecular assessment of cell phenotype that the overexpression of PPAR* did not alter the myogenic differentiation program of G8 myoblasts. We showed that PPAR* does not transactivate promoters containing PPARgamma-responsive elements but transactivates promoters containing PPAR*-responsive elements that are at least 80% identical to a 20-bp consensus. We improved the rosiglitazone-dependent gene switch by tuning PPAR* expression with a scaffold/matrix attachment region and by expressing both PPAR* and the reporter gene under the control of PPAR*-responsive elements. Treatment of cultured murine muscle cells (myotubes) with rosiglitazone induced reporter gene expression from assay background up to the level attained by a CMV I/E promoter-enhancer. These results indicate the potential of the PPAR* gene switch for use in gene therapy applications.
    Keywords: Gene Expression Regulation ; Muscle Fibers, Skeletal -- Metabolism ; Thiazolidinediones -- Pharmacology;
    ISSN: 1525-0016
    E-ISSN: 1525-0024
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  • 6
    In: International Journal of Oncology, 03/01/2001
    ISSN: 1019-6439
    E-ISSN: 1791-2423
    Source: CrossRef
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  • 7
    Language: English
    In: Molecular and Cellular Endocrinology, 2003, Vol.200(1), pp.9-18
    Description: In an attempt to determine the susceptibility of spermatogonia to malignant transformation transgenic mice were generated harboring a 1.3 kb 5'-flanking region of the germ cell specific expressed human testis specific protein, Y-encoded gene fused with the simian virus 40 large T antigen (TAg). Unexpectedly, TAg expression in transgenic mice was also detected in somatic tissues. Between days 65 and 85 after birth most of the transgenic mice developed anterior lobe tumors of the pituitary gland and to a less extent medulla type tumors of the adrenal gland. In addition, a few older transgenic mice developed tumors of the seminal vesicle, but no testicular tumors were observed in transgenic mice up to an age of 5 months. The pituitary tumors were immunoreactive for anti-prolactin (PRL) and anti-adrenocorticotropic hormone (ACTH). PRL and corticosterone concentrations in serum of transgenic mice were significantly increased. Taken together, our studies provide a novel mouse model for pituitary adenomas displaying a unique combination of hormone expression by tumor cells secreting PRL and ACTH.
    Keywords: Sv40 Large T-Antigen ; Transgenic Mice ; Ectopic Expression ; Pituitary Adenomas ; ACTH ; PRL ; Medicine ; Anatomy & Physiology
    ISSN: 0303-7207
    E-ISSN: 1872-8057
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  • 8
    Language: German
    Keywords: 570 Biowissenschaften ; Biologie ; Spermatogenese ; Maligne Transformation ; 42.13 ; 42.15 ; 42.23 ; W
    Source: Networked Digital Library of Theses and Dissertations
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