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Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes

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Abstract

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

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References

  1. Ogbourne, S., & Antalis, T. M. (1998). Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. The Biochemical Journal, 331(Pt 1), 1–14.

    CAS  Google Scholar 

  2. Struhl, K. (1999). Fundamentally different logic of gene regulation in eukaryotes and prokaryotes. Cell, 98, 1–4.

    Article  CAS  Google Scholar 

  3. Trinklein, N. D., Aldred, S. J., Saldanha, A. J., & Myers, R. M. (2003). Identification and functional analysis of human transcriptional promoters. Genome Research, 13, 308–312.

    Article  CAS  Google Scholar 

  4. Cormack, B. P., Valdivia, R. H., & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, 173, 33–38.

    Article  CAS  Google Scholar 

  5. Crivat, G., & Taraska, J. W. (2012). Imaging proteins inside cells with fluorescent tags. Trends in Biotechnology, 30, 8–16.

    Article  CAS  Google Scholar 

  6. Davey, H. M., & Kell, D. B. (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single-cell analyses. Microbiological Reviews, 60, 641–696.

    CAS  Google Scholar 

  7. Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K., & Tsien, R. Y. (2000). The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proceedings of the National Academy of Sciences of the United States of America, 97, 11990–11995.

    Article  CAS  Google Scholar 

  8. Chudakov, D. M., Lukyanov, S., & Lukyanov, K. A. (2005). Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology, 23, 605–613.

    Article  CAS  Google Scholar 

  9. Linn, T., & St Pierre, R. (1990). Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. Journal of Bacteriology, 172, 1077–1084.

    CAS  Google Scholar 

  10. Yu, X., Zhan, X., D’Costa, J., Tanavde, V. M., Ye, Z., Peng, T., et al. (2003). Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Molecular therapy: The journal of the American Society of Gene Therapy, 7, 827–838.

    Article  CAS  Google Scholar 

  11. Golding, M. C., & Mann, M. R. (2011). A bidirectional promoter architecture enhances lentiviral transgenesis in embryonic and extraembryonic stem cells. Gene Therapy, 18, 817–826.

    Article  CAS  Google Scholar 

  12. Low, K., Blesch, A., Herrmann, J., & Tuszynski, M. H. (2010). A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury. Gene Therapy, 17, 577–591.

    Article  CAS  Google Scholar 

  13. Finn, J., Lee, A. C., MacLachlan, I., & Cullis, P. (2004). An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression. Gene Therapy, 11, 276–283.

    Article  CAS  Google Scholar 

  14. Nettelbeck, D. M., Jerome, V., & Muller, R. (1999). A dual specificity promoter system combining cell cycle-regulated and tissue-specific transcriptional control. Gene Therapy, 6, 1276–1281.

    Article  CAS  Google Scholar 

  15. Andrianaki, A., Siapati, E. K., Hirata, R. K., Russell, D. W., & Vassilopoulos, G. (2010). Dual transgene expression by foamy virus vectors carrying an endogenous bidirectional promoter. Gene Therapy, 17, 380–388.

    Article  CAS  Google Scholar 

  16. D’Costa, J., Mansfield, S. G., & Humeau, L. M. (2009). Lentiviral vectors in clinical trials: Current status. Current Opinion in Molecular Therapeutics, 11, 554–564.

    Google Scholar 

  17. Lois, C., Hong, E. J., Pease, S., Brown, E. J., & Baltimore, D. (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 295, 868–872.

    Article  CAS  Google Scholar 

  18. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., et al. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 272, 263–267.

    Article  CAS  Google Scholar 

  19. Ramezani, A., Hawley, T. S., & Hawley, R. G. (2000). Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Molecular therapy: The journal of the American Society of Gene Therapy, 2, 458–469.

    Article  CAS  Google Scholar 

  20. Singer, O., Tiscornia, G., Ikawa, M., & Verma, I. M. (2006). Rapid generation of knockdown transgenic mice by silencing lentiviral vectors. Nature Protocols, 1, 286–292.

    Article  CAS  Google Scholar 

  21. Kay, M. A., Glorioso, J. C., & Naldini, L. (2001). Viral vectors for gene therapy: The art of turning infectious agents into vehicles of therapeutics. Nature Medicine, 7, 33–40.

    Article  CAS  Google Scholar 

  22. Cui, Y., Golob, J., Kelleher, E., Ye, Z., Pardoll, D., & Cheng, L. (2002). Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells. Blood, 99, 399–408.

    Article  CAS  Google Scholar 

  23. Pfeifer, A., Ikawa, M., Dayn, Y., & Verma, I. M. (2002). Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proceedings of the National Academy of Sciences of the United States of America, 99, 2140–2145.

    Article  CAS  Google Scholar 

  24. Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki, N., Miki, H., et al. (2004). Generation of pluripotent stem cells from neonatal mouse testis. Cell, 119, 1001–1012.

    Article  CAS  Google Scholar 

  25. Shamblott, M. J., Axelman, J., Wang, S., Bugg, E. M., Littlefield, J. W., Donovan, P. J., et al. (1998). Derivation of pluripotent stem cells from cultured human primordial germ cells. Proceedings of the National Academy of Sciences of the United States of America, 95, 13726–13731.

    Article  CAS  Google Scholar 

  26. Kaempfer, R., Rosen, H., & Israeli, R. (1978). Translational control: Recognition of the methylated 5′ end and an internal sequence in eukaryotic mRNA by the initiation factor that binds methionyl-tRNAfMet. Proceedings of the National Academy of Sciences of the United States of America, 75, 650–654.

    Article  CAS  Google Scholar 

  27. Devroe, E., & Silver, P. A. (2004). Therapeutic potential of retroviral RNAi vectors. Expert Opinion on Biological Therapy, 4, 319–327.

    Article  CAS  Google Scholar 

  28. Mao, G., Marotta, F., Yu, J., Zhou, L., Yu, Y., Wang, L., et al. (2008). DNA context and promoter activity affect gene expression in lentiviral vectors. Acta Bio-medica: Atenei Parmensis, 79, 192–196.

    CAS  Google Scholar 

  29. Tononi, G., Sporns, O., & Edelman, G. M. (1999). Measures of degeneracy and redundancy in biological networks. Proceedings of the National Academy of Sciences of the United States of America, 96, 3257–3262.

    Article  CAS  Google Scholar 

  30. Baup, D., Fraga, L., Pernot, E., Van Acker, A., Vanherck, A. S., Breckpot, K., et al. (2010). Variegation and silencing in a lentiviral-based murine transgenic model. Transgenic Research, 19, 399–414.

    Article  CAS  Google Scholar 

  31. Tian, J., & Andreadis, S. T. (2009). Independent and high-level dual-gene expression in adult stem-progenitor cells from a single lentiviral vector. Gene Therapy, 16, 874–884.

    Article  CAS  Google Scholar 

  32. Boulogne, B., Levacher, C., Durand, P., & Habert, R. (1999). Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: Immunolocalization and implication in the control of the number of gonocytes. Biology of Reproduction, 61, 1548–1557.

    Article  CAS  Google Scholar 

  33. Menke, D. B., Koubova, J., & Page, D. C. (2003). Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior-to-posterior wave. Developmental Biology, 262, 303–312.

    Article  CAS  Google Scholar 

  34. Oulad-Abdelghani, M., Bouillet, P., Decimo, D., Gansmuller, A., Heyberger, S., Dolle, P., et al. (1996). Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene. The Journal of Cell Biology, 135, 469–477.

    Article  CAS  Google Scholar 

  35. Yao, H. H., DiNapoli, L., & Capel, B. (2003). Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads. Development, 130, 5895–5902.

    Article  CAS  Google Scholar 

  36. Zhou, Q., Li, Y., Nie, R., Friel, P., Mitchell, D., Evanoff, R. M., et al. (2008). Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biology of Reproduction, 78, 537–545.

    Article  CAS  Google Scholar 

  37. Manova, K., & Bachvarova, R. F. (1991). Expression of c-kit encoded at the W locus of mice in developing embryonic germ cells and presumptive melanoblasts. Developmental Biology, 146, 312–324.

    Article  CAS  Google Scholar 

  38. Orr-Urtreger, A., Avivi, A., Zimmer, Y., Givol, D., Yarden, Y., & Lonai, P. (1990). Developmental expression of c-kit, a proto-oncogene encoded by the W locus. Development, 109, 911–923.

    CAS  Google Scholar 

  39. Zhang, L., Tang, J., Haines, C. J., Feng, H. L., Lai, L., Teng, X., et al. (2011). c-kit and its related genes in spermatogonial differentiation. Spermatogenesis, 1, 186–194.

    Article  Google Scholar 

  40. Adams, I. R., & McLaren, A. (2002). Sexually dimorphic development of mouse primordial germ cells: Switching from oogenesis to spermatogenesis. Development, 129, 1155–1164.

    CAS  Google Scholar 

  41. Shinohara, T., Avarbock, M. R., & Brinster, R. L. (1999). beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proceedings of the National Academy of Sciences of the United States of America, 96, 5504–5509.

    Article  CAS  Google Scholar 

  42. Shinohara, T., Orwig, K. E., Avarbock, M. R., & Brinster, R. L. (2000). Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proceedings of the National Academy of Sciences of the United States of America, 97, 8346–8351.

    Article  CAS  Google Scholar 

  43. Dann, C. T., Alvarado, A. L., Molyneux, L. A., Denard, B. S., Garbers, D. L., & Porteus, M. H. (2008). Spermatogonial stem cell self-renewal requires OCT4, a factor downregulated during retinoic acid-induced differentiation. Stem Cells, 26, 2928–2937.

    Article  CAS  Google Scholar 

  44. Maeda, K., Nishiyama, C., Ogawa, H., & Okumura, K. (2010). GATA2 and Sp1 positively regulate the c-kit promoter in mast cells. Journal of Immunology, 185, 4252–4260.

    Article  CAS  Google Scholar 

  45. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., et al. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147.

    Article  CAS  Google Scholar 

  46. Trichas, G., Begbie, J., & Srinivas, S. (2008). Use of the viral 2A peptide for bicistronic expression in transgenic mice. BMC Biology, 6, 40.

    Article  Google Scholar 

  47. Pellegrini, M., Filipponi, D., Gori, M., Barrios, F., Lolicato, F., Grimaldi, P., et al. (2008). ATRA and KL promote differentiation toward the meiotic program of male germ cells. Cell Cycle, 7, 3878–3888.

    Article  CAS  Google Scholar 

  48. Xu, X., Pantakani, D. V., Luhrig, S., Tan, X., Khromov, T., Nolte, J., et al. (2011). Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency. PLoS ONE, 6, e22413.

    Article  CAS  Google Scholar 

  49. Palmqvist, L., Glover, C. H., Hsu, L., Lu, M., Bossen, B., Piret, J. M., et al. (2005). Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells, 23, 663–680.

    Article  CAS  Google Scholar 

  50. Morley, P., & Whitfield, J. F. (1993). The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types. Journal of Cellular Physiology, 156, 219–225.

    Article  CAS  Google Scholar 

  51. Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2, 905–909.

    Article  CAS  Google Scholar 

  52. Alimperti, S., Lei, P., Tian, J., & Andreadis, S. T. (2012). A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation. Gene Therapy, 19, 1123–1132.

    Article  CAS  Google Scholar 

  53. Conrad, S., Renninger, M., Hennenlotter, J., Wiesner, T., Just, L., Bonin, M., et al. (2008). Generation of pluripotent stem cells from adult human testis. Nature, 456, 344–349.

    Article  CAS  Google Scholar 

  54. Geijsen, N., & Jones, D. L. (2008). Seminal discoveries in regenerative medicine: Contributions of the male germ line to understanding pluripotency. Human Molecular Genetics, 17, R16–R22.

    Article  CAS  Google Scholar 

  55. Guan, K., Nayernia, K., Maier, L. S., Wagner, S., Dressel, R., Lee, J. H., et al. (2006). Pluripotency of spermatogonial stem cells from adult mouse testis. Nature, 440, 1199–1203.

    Article  CAS  Google Scholar 

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Acknowledgments

We are very grateful to Dr. Corrine Kostic (University of Lausanne, Switzerland) for their valuable discussions and to Ali Fallah and Muhammad Irfan-maqsood for their technical assistance and critical reading of this manuscript. This study was supported by the Grant no. 27672 from Ferdowsi University of Mashhad.

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, Ferdowsi University of Mashhad , Mashhad, Iran

    Mahtab Dastpak, Maryam M. Matin, Moein Farshchian, Sajjad Sisakhtnezhad, Sohrab Boozarpour & Ahmad Reza Bahrami

  2. Cell and Molecular Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran

    Mahtab Dastpak, Maryam M. Matin, Moein Farshchian, Madjid Momeni-Moghaddam, Sajjad Sisakhtnezhad, Sohrab Boozarpour & Ahmad Reza Bahrami

  3. Unit of Gene Therapy and Stem Cell Biology, Service of Ophthalmology, Jules-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland

    Yvan Arsenijevic

  4. Department of Stem Cells and Regenerative Medicine, ACECR, Mashhad, Iran

    Hamid Reza Bidkhori & Mahdi Mirahmadi

  5. Department of Biology, Faculty of Sciences, Hakim Sabzevari University, Sabzevar, Iran

    Madjid Momeni-Moghaddam

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  1. Mahtab Dastpak
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Correspondence to Ahmad Reza Bahrami.

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Dastpak, M., Matin, M.M., Farshchian, M. et al. Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes. Mol Biotechnol 56, 1100–1109 (2014). https://doi.org/10.1007/s12033-014-9790-9

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