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Pitx3 promoter directs Cre-recombinase specifically in a human neuroblastoma cell line

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Abstract

The Pitx3 gene is a homeobox transcription factor. This gene is expressed only in midbrain dopaminergic-neurons in the central nervous system, where its expression is important for development and survival of mesencephalic-dopaminergic neurons. The promoter region of the Pitx3 gene is not yet completely delimited. We used the Cre-loxP system to demonstrate the efficiency and specificity of a 4.2-kbp sequence in the 5′-flanking region of the Pitx3-gene promoter inserted in the 5′-terminus of the Cre-recombinase gene. A Cre-recombinase-reporter assay indicated that this 5′-flanking region possesses promoter activity. The cell-specific gene regulation of the Pitx3 promoter in neurons was demonstrated by a reverse-transcription polymerase chain reaction (RT-PCR) and Western blot detection of Cre-recombinase in SH-SY5Y cells but not in MCF7 cells. Functionality of the Cre-recombinase was confirmed in vitro. The Pitx3-promoter-Cre cassette here described can be used to develop transgenic mice with the specific expression of Cre-recombinase in midbrain-dopaminergic neurons to elucidate the gene function in which the conventional knockout leads to an early lethal phenotype. Such specific expression of the Pitx3 promoter may be used for gene therapy studies of Parkinson’s disease.

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References

  1. Castillo SO, Baffi JS, Palkovits M, Goldstein DS, Kopin IJ, Witta J, Magnuson MA, Nikodem VM (1998) Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene. Mol Cell Neurosci 11:36–46

    Article  PubMed  CAS  Google Scholar 

  2. Saucedo-Cardenas O, Quintana-Hau JD, Le WD, Smidt MP, Cox JJ, De Mayo F, Burbach JP, Conneely OM (1998) Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci USA 95:4013–4018

    Article  PubMed  CAS  Google Scholar 

  3. Zetterstrom RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann T (1997) Dopamine neuron agenesis in Nurr1-deficient mice. Science 276:248–250

    Article  PubMed  CAS  Google Scholar 

  4. Nunes I, Tovmasian LT, Silva RM, Burke RE, Goff SP (2003) Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci USA 100:4245–4250

    Article  PubMed  CAS  Google Scholar 

  5. Smidt MP, Smits SM, Bouwmeester H, Hamers FP, Van der Linden AJ, Hellemons AJ, Graw J, Burbach JP (2004) Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. Development 131:1145–1155

    Article  PubMed  CAS  Google Scholar 

  6. Smidt MP, Smits SM, Burbach JP (2004) Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra. Cell Tissue Res 318:35–43

    Article  PubMed  CAS  Google Scholar 

  7. Wang X, Li X, Wang K, Zhou H, Xue B, Li L, Wang X (2004) Forskolin cooperating with growth factor on generation of dopaminergic neurons from human fetal mesencephalic neural progenitor cells. Neurosci Lett 362:117–121

    Article  PubMed  CAS  Google Scholar 

  8. Van den Munckhof P, Luk KC, Ste-Marie L, Montgomery J, Blanchet PJ, Sadikot AF, Drouin J (2003) Pitx3 is required for motor activity and for survival of a subset of midbrain dopaminergic neurons. Development 130:2535–2542

    Article  PubMed  Google Scholar 

  9. Semina EV, Murray JC, Reiter R, Hrstka RF, Graw J (2000) Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice. Hum Mol Genet 9:1575–1585

    Article  PubMed  CAS  Google Scholar 

  10. Hwang DY, Ardayfio P, Kang UJ, Semina EV, Kim KS (2003) Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Mol Brain Res 114:123–131

    Article  PubMed  CAS  Google Scholar 

  11. Smidt MP, Van Schaick HS, Lanctot C, Tremblay JJ, Cox JJ, Van der Kleij AA, Wolterink G, Drouin J, Burbach JP (1997) A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons. Proc Natl Acad Sci USA 94:13305–13310

    Article  PubMed  CAS  Google Scholar 

  12. Quentien MH, Manfroid I, Moncet D, Gunz G, Muller M, Grino M, Enjalbert A, Pellegrini I (2002) Pitx factors are involved in basal and hormone-regulated activity of the human prolactin promoter. J Biol Chem 277:44408–44416

    Article  PubMed  CAS  Google Scholar 

  13. Quentien MH, Pitoia F, Gunz G, Guillet MP, Enjalbert A, Pellegrini I (2002) Regulation of prolactin, GH, and Pit-1 gene expression in anterior pituitary by Pitx2: an approach using Pitx2 mutants. Endocrinology 143:2839–2851

    Article  PubMed  CAS  Google Scholar 

  14. Quirk CC, Lozada KL, Keri RA, Nilson JH (2001) A single Pitx1 binding site is essential for activity of the LHbeta promoter in transgenic mice. Mol Endocrinol 15:734–746

    Article  PubMed  CAS  Google Scholar 

  15. Rieger DK, Reichenberger E, McLean W, Sidow A, Olsen BR (2001) A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice. Genomics 72:61–72

    Article  PubMed  CAS  Google Scholar 

  16. Grimm C, Chatterjee B, Favor J, Immervoll T, Loster J, Klopp N, Sandulache R, Graw J (1998) Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered Pax6 and Six3 gene expression pattern. Dev Genet 23:299–316

    Article  PubMed  CAS  Google Scholar 

  17. Hwang DY, Fleming SM, Ardayfio P, Moran-Gates T, Kim H, Tarazi FI, Chesselet MF, Kim KS (2005) 3,4-dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson’s disease. J Neurosci 25:2132–2137

    Article  PubMed  CAS  Google Scholar 

  18. Hoess RH, Ziese M, Sternberg N (1982) P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc Natl Acad Sci USA 79:3398–3402

    Article  PubMed  CAS  Google Scholar 

  19. Mangoura D, Theofilopoulos S, Karouzaki S, Tsirimonaki E (2006) 12-O-tetradecanoyl-phorbol-13-acetate-dependent up-regulation of dopaminergic gene expression requires Ras and neurofibromin in human IMR-32 neuroblastoma. J Neurochem 97:97–103

    Article  PubMed  CAS  Google Scholar 

  20. McLaughlin D, Tsirimonaki E, Vallianatos G, Sakellaridis N, Chatzistamatiou T, Stavropoulos-Gioka C, Tsezou A, Messinis I, Mangoura D (2006) Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells. J Neurosci Res 83:1190–1200

    Article  PubMed  CAS  Google Scholar 

  21. Zhao S, Maxwell S, Jimenez-Beristain A, Vives J, Kuehner E, Zhao J, O’Brien C, de Felipe C, Semina E, Li M (2004) Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur J Neurosci 19:1133–1140

    Article  PubMed  Google Scholar 

  22. Sacchetti P, Brownschidle LA, Granneman JG, Bannon MJ (1999) Characterization of the 5′-flanking region of the human dopamine transporter gene. Brain Res Mol Brain Res 74:167–174

    Article  PubMed  CAS  Google Scholar 

  23. Kessler MA, Yang M, Gollomp KL, Jin H, Iacovitti L (2003) The human tyrosine hydroxylase gene promoter. Brain Res Mol Brain Res 112:8–23

    Article  PubMed  CAS  Google Scholar 

  24. Lin LF, Doherty DH, Lile JD, Becktesh S, Collins F (1993) GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260:1130–1132

    Article  PubMed  CAS  Google Scholar 

  25. Lindholm P, Voutilainen MH, Laurén J, Peranen J, Leppanen VM, Andressoo JO, Lindahl M, Janhunen S, Kalkkinen N, Timmusk T, Tuominen RK, Saarma M (2007) Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature 448:73–77

    Article  PubMed  CAS  Google Scholar 

  26. Gonzalez-Barrios JA, Lindahl M, Bannon MJ, Anaya-Martínez V, Flores G, Navarro-Quiroga I, Trudeau LE, Aceves J, Martinez-Arguelles DB, García-Villegas R, Jimenez I, Segovia J, Martínez-Fong D (2006) Neurotensin Polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats. Mol Ther 14:857–865

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by National Council of Science and Technology (CONACYT) of Mexico, Grant 41616 to O.S-C. DL C-C and H.R-R are recipients of a fellowship from CONACYT of Mexico. Thanks to Dr Ellis Glazier for editing this English-language text.

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Authors and Affiliations

  1. Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, NL, Mexico

    Diana L. Castillo-Carranza, Humberto Rodríguez-Rocha, Roberto Montes-de-Oca-Luna, Julio Sepúlveda-Saavedra & Odila Saucedo-Cárdenas

  2. División de Genética, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social (IMSS), Col. Independencia, Monterrey, NL, 64720, Mexico

    Humberto Rodríguez-Rocha & Odila Saucedo-Cárdenas

  3. Servicio de Neurología, Hospital Universitario, Universidad Autónoma de Nuevo León (UANL), Monterrey, NL, Mexico

    Héctor R. Martínez

  4. Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico, DF, Mexico

    Yolanda López-Vidal

Authors
  1. Diana L. Castillo-Carranza
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  2. Humberto Rodríguez-Rocha
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  3. Roberto Montes-de-Oca-Luna
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  6. Yolanda López-Vidal
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  7. Odila Saucedo-Cárdenas
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Correspondence to Odila Saucedo-Cárdenas.

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Castillo-Carranza, D.L., Rodríguez-Rocha, H., Montes-de-Oca-Luna, R. et al. Pitx3 promoter directs Cre-recombinase specifically in a human neuroblastoma cell line. Mol Cell Biochem 309, 223–227 (2008). https://doi.org/10.1007/s11010-007-9655-7

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  • DOI: https://doi.org/10.1007/s11010-007-9655-7

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