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volume 46 issue 1 (march 2012) : 15-21

MOLECULAR AND EPIGENETIC STUDY OF H19 GENE IN GOAT (CAPRA HIRCUS)

S
S.V. Lal
S
S. Singh
R
R. Kumari1
S
S. Kumar2
1Biotechnology Centre, J.N. Krishi Vishwavidyalaya, Jabalpur-482 004, India

  • Submitted|

  • First Online |

  • doi

Cite article:- Lal S.V., Singh S., Kumari1 R., Kumar2 S. (2025). MOLECULAR AND EPIGENETIC STUDY OF H19 GENE IN GOAT (CAPRA HIRCUS). Indian Journal of Animal Research. 46(1): 15-21. doi: .

ABSTRACT

Due to the high incidence of abnormalities and the inefficiency of generating goat kids through somatic cell nuclear transfer (SCNT), the development of a model system in goat to investigate potential problems is warranted. In nuclear transfer, where genomic imprinting has been implicated as a major cause for these problems, epigenetic regulation of developmentally important genes may give us the clue regarding the probable reasons for the low efficiency observed in SCNT. H19 is one such paternally imprinted gene. The present investigation was undertaken to study the methylation status of CTCF III binding region in the upstream of H19 gene before and after reprogramming by serum starvation method, in cultured fibroblast cells. Fibroblast cells were cultured up to sixth passage and genomic DNA was extracted before and after reprogramming. Genomic DNA samples were then used to amplify 295 bp fragment of H19 CTCF III binding region. The nucleotide sequence identified in this fragment had 19 CpG motifs. Genomic DNA samples were then treated with sodium bisulphite to analyse the methylation status of identified CpG motifs. The bisulphite converted genomic DNA was amplified by bisulphite sequencing primer (BSP) set. The amplified fragments of bisulphite converted genomic DNA samples of reprogrammed and non-reprogrammed cells were then sequenced. Variation in the sequences were obtained from bisulphite converted genomic DNA of reprogrammed and non-reprogrammed cells. The nucleotide sequence analysis of bisulphite converted cultured non-reprogrammed cells revealed methylation of 6 CpG motifs. The level of methylation observed in the study for 295 bp gene fragment was about 31.5%.  However, in reprogrammed cells the CpG motifs were found to be unmethylated. Therefore, it is concluded that a reduction in the level of methylation was observed in reprogrammed fibroblast cells after serum starvation.

REFERENCES

  1. Ariel, I., Ayesh S., Perlman E.J., Pizov G., Tanos V., Schneider T., Erdmann V.A., Podeh D., Komitowski D., Quasem A.S., De Groot, Hochberg A. (1997). The product of the imprinted H19 gene is an oncofetal RNA. Molec. Path., 50: 34-44.
  2. Berg, D. K., Li C., Asher G., D.N. Wells and Oback B. (2007). Red deer cloned from antler stem cells and their differentiated progeny. Biol. Reprod. 77(3): 384-94.
  3. Binder, G., A. K. Seidel, K. Weber, M. Haase, H. A. Wollmann, M. B. Ranke (2006). IGF-II Serum level are normal in childrens with Silver Russel syndrome who frequently carry epimutation of at the IGFII locus. Jour. Clin. Endrocrinol. Metabo., 91: 1402-1407.
  4. Cezar, G.G. (2003). Epigenetic reprogramming of cloned animals. Cloning Stem Cells, 5: 165-180..
  5. Eversole-Cire, P., Anne C. Ferguson-Smith, M. Azim Surani, and Peter A. Jones (1995). Coordinate Regulation of Igf-2 and H19 in Cultured Cells. Cell Growth and Differentiation, 337:337-345.
  6. Feil, R., J. Charlton, A.P. Bird, J. Walter and W. Reik (1994). Methylation analysis of individual chromosomes : Improved protocol for bisulphite genomic sequencing. Nucleic Acids Res., 22 : 695-696
  7. Fujita, N. and P.A. Wade (2004). Nuclear transfer: epigenetics plays a visit, Nat cell Biol., 10:984-990.
  8. Giraldo, M. Angelica, Darin A. Hylan, Casey B. Ballard, Megan N. Purpera, Todd D. Vaught, John W. Lynn, Robert A. Godke, and Kenneth R. Bondioli (2008). Effect of epigenetic modifications of Donor somatic cells on the subsequent chromatin remodeling of cloned bovine embryos. Biol. Reprod., 78: 832-840
  9. Jie, C., Li Dong Jie, Liu YanQin, Zhang Cui, Dai YunPing, Li SheJie, Li Ning (2008). DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines. Chinese Science Bulletin, 53: 1996-2011.
  10. Keefer, C., R. Keyston, A. Lazaris, B. Bhatia, I. Begin, A. Bilodeau, F. Zhou, N. Kafidi, B. Wang, H. Baldassarre, C. Karatzas (2002). Production of cloned goats after nuclear transfer using adult somatic cells. Biol. Reprod., 66:199-203.
  11. Khatib, H. and V. Schutzkus (2006). The expression profile of the H19 gene in cattle. Mamm Genome., 17(9) : 991-996
  12. Li, L.H. and R. Dahiya (2002). MethPrimer: designing primers for methylation PCRs. Bioinformatics, 11: 1427-1431.
  13. Paoloni- Giacobino, A and J.R. Chaillet (2004). Genomic imprinting and assisted reproduction. Reprod. Health, 1(1):6
  14. Paulsen, M., S. Takada, N.A. Youngson, M. Benchaib, C. Charlier, K. Segers, M. Georges and Anne C. F. Smith (2001). Comparative Sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2- H19 region. Genome Res, 11(12) : 2085-2094
  15. Rice, J.C., C.D. Allis (2001). Code of silence. Nature, 414:258-261.
  16. Sambrook, J., and D. W. Russel (2001).Molecular cloning : A laboratory Manual.Cold spring harbour laboaory press,New york, 2: 8.18-8.25.
  17. Thurston, A., Jane Taylor, John Gardner, Kevin D Sinclair and Lorraine E Young (2008). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction, 135: 29-40.
  18. Wilmut, I., A.E. Schnieke, J. McWhir, A.J. Kind, K.H. Campbell (1997). Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810-813.
  19. Yang, M and X. Tian (2005). Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones. Mol. Reprod. Dev., 71: 431-438.
  20. Yang, F., R. Hao, B. Kessler, G. Brem, E. Wolf and V. Zakhartchenko (2007).Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction, 133(1): 219-30.
  21. Young, L.E, E. Angelika, Schnieke, J. Kenneth, McCreath, Wieckowski, Galia Konfortova, Kenneth Fernandes, Grazyna Ptak, Alex J. Kind, Ian Wilmut, Pasqualino Loi and Robert Feil (2003). Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer. Mechanisms of Development, 120: 1433–1442.
  22. Zhang, Y., Thomas Shields, Taria Crenshaw, Yue Hao, Thomas Moulton and Benjamin Tycko (1993). Imprinting of human H19: Allele-specific CpG methylation, loss of the active allele in Wilms Tumor, and potential for somatic allele switching. American Journal of Human Genetics, 53: 113-124.
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Indian Journal of Animal Research
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    volume 46 issue 1 (march 2012) : 15-21

    MOLECULAR AND EPIGENETIC STUDY OF H19 GENE IN GOAT (CAPRA HIRCUS)

    S
    S.V. Lal
    S
    S. Singh
    R
    R. Kumari1
    S
    S. Kumar2
    1Biotechnology Centre, J.N. Krishi Vishwavidyalaya, Jabalpur-482 004, India

    • Submitted|

    • First Online |

    • doi

    Cite article:- Lal S.V., Singh S., Kumari1 R., Kumar2 S. (2025). MOLECULAR AND EPIGENETIC STUDY OF H19 GENE IN GOAT (CAPRA HIRCUS). Indian Journal of Animal Research. 46(1): 15-21. doi: .

    ABSTRACT

    Due to the high incidence of abnormalities and the inefficiency of generating goat kids through somatic cell nuclear transfer (SCNT), the development of a model system in goat to investigate potential problems is warranted. In nuclear transfer, where genomic imprinting has been implicated as a major cause for these problems, epigenetic regulation of developmentally important genes may give us the clue regarding the probable reasons for the low efficiency observed in SCNT. H19 is one such paternally imprinted gene. The present investigation was undertaken to study the methylation status of CTCF III binding region in the upstream of H19 gene before and after reprogramming by serum starvation method, in cultured fibroblast cells. Fibroblast cells were cultured up to sixth passage and genomic DNA was extracted before and after reprogramming. Genomic DNA samples were then used to amplify 295 bp fragment of H19 CTCF III binding region. The nucleotide sequence identified in this fragment had 19 CpG motifs. Genomic DNA samples were then treated with sodium bisulphite to analyse the methylation status of identified CpG motifs. The bisulphite converted genomic DNA was amplified by bisulphite sequencing primer (BSP) set. The amplified fragments of bisulphite converted genomic DNA samples of reprogrammed and non-reprogrammed cells were then sequenced. Variation in the sequences were obtained from bisulphite converted genomic DNA of reprogrammed and non-reprogrammed cells. The nucleotide sequence analysis of bisulphite converted cultured non-reprogrammed cells revealed methylation of 6 CpG motifs. The level of methylation observed in the study for 295 bp gene fragment was about 31.5%.  However, in reprogrammed cells the CpG motifs were found to be unmethylated. Therefore, it is concluded that a reduction in the level of methylation was observed in reprogrammed fibroblast cells after serum starvation.

    REFERENCES

    1. Ariel, I., Ayesh S., Perlman E.J., Pizov G., Tanos V., Schneider T., Erdmann V.A., Podeh D., Komitowski D., Quasem A.S., De Groot, Hochberg A. (1997). The product of the imprinted H19 gene is an oncofetal RNA. Molec. Path., 50: 34-44.
    2. Berg, D. K., Li C., Asher G., D.N. Wells and Oback B. (2007). Red deer cloned from antler stem cells and their differentiated progeny. Biol. Reprod. 77(3): 384-94.
    3. Binder, G., A. K. Seidel, K. Weber, M. Haase, H. A. Wollmann, M. B. Ranke (2006). IGF-II Serum level are normal in childrens with Silver Russel syndrome who frequently carry epimutation of at the IGFII locus. Jour. Clin. Endrocrinol. Metabo., 91: 1402-1407.
    4. Cezar, G.G. (2003). Epigenetic reprogramming of cloned animals. Cloning Stem Cells, 5: 165-180..
    5. Eversole-Cire, P., Anne C. Ferguson-Smith, M. Azim Surani, and Peter A. Jones (1995). Coordinate Regulation of Igf-2 and H19 in Cultured Cells. Cell Growth and Differentiation, 337:337-345.
    6. Feil, R., J. Charlton, A.P. Bird, J. Walter and W. Reik (1994). Methylation analysis of individual chromosomes : Improved protocol for bisulphite genomic sequencing. Nucleic Acids Res., 22 : 695-696
    7. Fujita, N. and P.A. Wade (2004). Nuclear transfer: epigenetics plays a visit, Nat cell Biol., 10:984-990.
    8. Giraldo, M. Angelica, Darin A. Hylan, Casey B. Ballard, Megan N. Purpera, Todd D. Vaught, John W. Lynn, Robert A. Godke, and Kenneth R. Bondioli (2008). Effect of epigenetic modifications of Donor somatic cells on the subsequent chromatin remodeling of cloned bovine embryos. Biol. Reprod., 78: 832-840
    9. Jie, C., Li Dong Jie, Liu YanQin, Zhang Cui, Dai YunPing, Li SheJie, Li Ning (2008). DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines. Chinese Science Bulletin, 53: 1996-2011.
    10. Keefer, C., R. Keyston, A. Lazaris, B. Bhatia, I. Begin, A. Bilodeau, F. Zhou, N. Kafidi, B. Wang, H. Baldassarre, C. Karatzas (2002). Production of cloned goats after nuclear transfer using adult somatic cells. Biol. Reprod., 66:199-203.
    11. Khatib, H. and V. Schutzkus (2006). The expression profile of the H19 gene in cattle. Mamm Genome., 17(9) : 991-996
    12. Li, L.H. and R. Dahiya (2002). MethPrimer: designing primers for methylation PCRs. Bioinformatics, 11: 1427-1431.
    13. Paoloni- Giacobino, A and J.R. Chaillet (2004). Genomic imprinting and assisted reproduction. Reprod. Health, 1(1):6
    14. Paulsen, M., S. Takada, N.A. Youngson, M. Benchaib, C. Charlier, K. Segers, M. Georges and Anne C. F. Smith (2001). Comparative Sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2- H19 region. Genome Res, 11(12) : 2085-2094
    15. Rice, J.C., C.D. Allis (2001). Code of silence. Nature, 414:258-261.
    16. Sambrook, J., and D. W. Russel (2001).Molecular cloning : A laboratory Manual.Cold spring harbour laboaory press,New york, 2: 8.18-8.25.
    17. Thurston, A., Jane Taylor, John Gardner, Kevin D Sinclair and Lorraine E Young (2008). Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage. Reproduction, 135: 29-40.
    18. Wilmut, I., A.E. Schnieke, J. McWhir, A.J. Kind, K.H. Campbell (1997). Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810-813.
    19. Yang, M and X. Tian (2005). Expression of imprinted genes is aberrant in deceased newborn cloned calves and relatively normal in surviving adult clones. Mol. Reprod. Dev., 71: 431-438.
    20. Yang, F., R. Hao, B. Kessler, G. Brem, E. Wolf and V. Zakhartchenko (2007).Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation. Reproduction, 133(1): 219-30.
    21. Young, L.E, E. Angelika, Schnieke, J. Kenneth, McCreath, Wieckowski, Galia Konfortova, Kenneth Fernandes, Grazyna Ptak, Alex J. Kind, Ian Wilmut, Pasqualino Loi and Robert Feil (2003). Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer. Mechanisms of Development, 120: 1433–1442.
    22. Zhang, Y., Thomas Shields, Taria Crenshaw, Yue Hao, Thomas Moulton and Benjamin Tycko (1993). Imprinting of human H19: Allele-specific CpG methylation, loss of the active allele in Wilms Tumor, and potential for somatic allele switching. American Journal of Human Genetics, 53: 113-124.
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