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Current IssueEditorial BoardOnline First ArticlesArchive

volume 49 issue 1 (february 2015) : 14-19,   Doi: 10.5958/0976-0555.2015.00003.5

Optimization of buffalo spermatozoa transfection with pbIFN-tau-EGFP gene by simple incubation technique

A
Arvind Kumar
N
N. Anand Laxmi*
K
Kuldeep Rai
A
Ashok Bainda
1DCP Division, National Dairy Research Institute, Karnal-132001, India.
Cite article:- Kumar Arvind, Laxmi* Anand N., Rai Kuldeep, Bainda Ashok (2025). Optimization of buffalo spermatozoa transfection with pbIFN-tau-EGFP gene by simple incubation technique. Indian Journal of Animal Research. 49(1): 14-19. doi: 10.5958/0976-0555.2015.00003.5.

ABSTRACT

The present study was conducted to optimize and analyze the uptake of pbIFN-tau-EGFP by spermatozoa of Murrah buffaloes (Bubalus bubalis) by simple incubation technique. Fresh semen samples from two Murrah buffaloes were selected based on mass activity (+3 and above) from the herd. Three different variables i.e. bovine Interferon tau gene conjugated to enhanced green fluorescent gene ( pbIFN-tau-EGFP) gene concentration, incubation time and number of spermatozoa, were considered for optimization of spermatozoa transfection by simple incubation technique. Before and after transfection, the spermatozoa samples were evaluated for sperm functional parameters, motility and viability. Spermatozoa incubated with different concentrations (10 and 20µg/ml) of  gene (pbIFN tau-EGFP) and without gene which served as control were subjected to  in vitro fertilization (IVF). A significant decrease (P

REFERENCES

  1. Alderson J., Wilson B., Laible G., Pfeffer P. and Huillier, P. (2006). Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis. Anim. Reprod. Sci., 91: 23–30.
  2. Anzar M. and Buhr M.M. (2006). Spontaneous uptake of exogenous DNA by bull spermatozoa. Therio., 65: 683-690.
  3. Bacci M.L., Galeat G., Mattoili M., Boni R. and Seren E. (1991) . In vitro maturation and in vitro fertilization of buffalo oocytes. In: Proceedings of the Third World Buffalo Congress, 1991, Varna, Bulgaria. Varna, Bulgaria: WBC. v. 3, pp. 599-603.
  4. Brackett B.G., Baranska W., Sawicki W. and Koprowski H. (1971). Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci.USA., 68:353-357
  5. Camaioni A., Russo M. A., Odorisio T., Gandolfi F., Fazio V.M. and Siracusa G. (1992). Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads. J ReprodFertil., 96: 203–212.
  6. Canovas S., Adan A.G. and Gadea J. (2010). Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer technique. Mol. Reprod. Dev., 77: 687-698.
  7. Chauhan M. S., Singla S.K., Palta P., Manik R.S. and Madan M.L. (1998). In vitro maturation and fertilization, and subsequent development of buffalo embryos: effects of oocyte quality and type of serum. Reprod. Fertil. Dev.,10 :173-177.
  8. Gagné M.B., Pothier F. and Sirard M.A. (1991). Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev. 29:6-15.
  9. Maione B., Lavitrano M., Spadafora C. and Kiessling A. A. (1998). Sperm mediated gene transfer in mice. Mol. Reprod. Dev., 50:406–409.
  10. Mortimer D. (1994) Practical Laboratory Andrology. Oxford UniversityPress, New York, USA. pp 66-69.
  11. Sambrook J. and Russell D.W. “Commonly Used Techniques in Molecular Cloning,” Appendix 8, in Molecular Cloning, Volume 3, 3rd edition, by Joseph Sambrook and David W. Russell. 2001. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA,
  12. Schellander K., Peli J., Schmall F., and Brem G., (1995).Artificial insemination in cattle with DNA-treated sperm. Anim. Biotechnol., 6:41-50.
  13. Shemesh M., Gurevich M., Harel-Markowitz E., Benvenisti L., Shore L.S. and Stram Y. (2000). Gene integration into bovine sperm genome and its expression in transgenic offspring. Mol. Reprod. Dev., 56:306–308.
  14. Smith K. and Spadafora C. (2005). Sperm-mediated gene transfer: applications and implications. Bioessays, 27: 551-562.
  15. Spadafora C. (1998). Sperm cells and foreign DNA: a controversial relation. Bioessays,20 : 955–964.
  16. Zani M., Lavitrano M., French D., Lulli V., Maione B., Sperandio S. and Spadafora C. (1995). The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp. Cell Res., 217: 57-64.
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    volume 49 issue 1 (february 2015) : 14-19,   Doi: 10.5958/0976-0555.2015.00003.5

    Optimization of buffalo spermatozoa transfection with pbIFN-tau-EGFP gene by simple incubation technique

    A
    Arvind Kumar
    N
    N. Anand Laxmi*
    K
    Kuldeep Rai
    A
    Ashok Bainda
    1DCP Division, National Dairy Research Institute, Karnal-132001, India.
    Cite article:- Kumar Arvind, Laxmi* Anand N., Rai Kuldeep, Bainda Ashok (2025). Optimization of buffalo spermatozoa transfection with pbIFN-tau-EGFP gene by simple incubation technique. Indian Journal of Animal Research. 49(1): 14-19. doi: 10.5958/0976-0555.2015.00003.5.

    ABSTRACT

    The present study was conducted to optimize and analyze the uptake of pbIFN-tau-EGFP by spermatozoa of Murrah buffaloes (Bubalus bubalis) by simple incubation technique. Fresh semen samples from two Murrah buffaloes were selected based on mass activity (+3 and above) from the herd. Three different variables i.e. bovine Interferon tau gene conjugated to enhanced green fluorescent gene ( pbIFN-tau-EGFP) gene concentration, incubation time and number of spermatozoa, were considered for optimization of spermatozoa transfection by simple incubation technique. Before and after transfection, the spermatozoa samples were evaluated for sperm functional parameters, motility and viability. Spermatozoa incubated with different concentrations (10 and 20µg/ml) of  gene (pbIFN tau-EGFP) and without gene which served as control were subjected to  in vitro fertilization (IVF). A significant decrease (P

    REFERENCES

    1. Alderson J., Wilson B., Laible G., Pfeffer P. and Huillier, P. (2006). Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis. Anim. Reprod. Sci., 91: 23–30.
    2. Anzar M. and Buhr M.M. (2006). Spontaneous uptake of exogenous DNA by bull spermatozoa. Therio., 65: 683-690.
    3. Bacci M.L., Galeat G., Mattoili M., Boni R. and Seren E. (1991) . In vitro maturation and in vitro fertilization of buffalo oocytes. In: Proceedings of the Third World Buffalo Congress, 1991, Varna, Bulgaria. Varna, Bulgaria: WBC. v. 3, pp. 599-603.
    4. Brackett B.G., Baranska W., Sawicki W. and Koprowski H. (1971). Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc. Natl. Acad. Sci.USA., 68:353-357
    5. Camaioni A., Russo M. A., Odorisio T., Gandolfi F., Fazio V.M. and Siracusa G. (1992). Uptake of exogenous DNA by mammalian spermatozoa: specific localization of DNA on sperm heads. J ReprodFertil., 96: 203–212.
    6. Canovas S., Adan A.G. and Gadea J. (2010). Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer technique. Mol. Reprod. Dev., 77: 687-698.
    7. Chauhan M. S., Singla S.K., Palta P., Manik R.S. and Madan M.L. (1998). In vitro maturation and fertilization, and subsequent development of buffalo embryos: effects of oocyte quality and type of serum. Reprod. Fertil. Dev.,10 :173-177.
    8. Gagné M.B., Pothier F. and Sirard M.A. (1991). Electroporation of bovine spermatozoa to carry foreign DNA in oocytes. Mol. Reprod. Dev. 29:6-15.
    9. Maione B., Lavitrano M., Spadafora C. and Kiessling A. A. (1998). Sperm mediated gene transfer in mice. Mol. Reprod. Dev., 50:406–409.
    10. Mortimer D. (1994) Practical Laboratory Andrology. Oxford UniversityPress, New York, USA. pp 66-69.
    11. Sambrook J. and Russell D.W. “Commonly Used Techniques in Molecular Cloning,” Appendix 8, in Molecular Cloning, Volume 3, 3rd edition, by Joseph Sambrook and David W. Russell. 2001. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA,
    12. Schellander K., Peli J., Schmall F., and Brem G., (1995).Artificial insemination in cattle with DNA-treated sperm. Anim. Biotechnol., 6:41-50.
    13. Shemesh M., Gurevich M., Harel-Markowitz E., Benvenisti L., Shore L.S. and Stram Y. (2000). Gene integration into bovine sperm genome and its expression in transgenic offspring. Mol. Reprod. Dev., 56:306–308.
    14. Smith K. and Spadafora C. (2005). Sperm-mediated gene transfer: applications and implications. Bioessays, 27: 551-562.
    15. Spadafora C. (1998). Sperm cells and foreign DNA: a controversial relation. Bioessays,20 : 955–964.
    16. Zani M., Lavitrano M., French D., Lulli V., Maione B., Sperandio S. and Spadafora C. (1995). The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake. Exp. Cell Res., 217: 57-64.
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