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Production of transgenic rabbit embryos through intracytoplasmic sperm injection

  • Qiuyan Li (a1), Jian Hou (a1), Sheng Wang (a1), Yongfu Chen (a1) and Xiao-Rong An (a2)...

Summary

The objective of this study was to test if intracytoplasmic sperm injection (ICSI)-mediated gene transfer was an effective method in the production of transgenic rabbit embryos. Rabbit sperm diluted in different media with various pH were treated by freezing without cryoprotectant, and their ability for DNA uptake was determined. In these experiments using production of transgenic rabbit embryos by ICSI, exogenous genes at three concentrations and of two conformation types were used. The rate of DNA association to the sperm seen by rhodamine-tagged DNA encoding green fluorescent protein (GFP) was 90.0%, 92.7%, 91.0%, 91.7%, and 92.3%, respectively in TCM199, DM, DPBS, CZB, and HCZB media. The DNA attachment to sperm was not affected by media pH within the range of 5.4–9.4 (p > 0.05). Expression of GFP first occurred at the 2-cell stage and continued to blastocyst formation. DNA concentration (between 5, 10, and 20 ng/μl) or conformation (linear and circular) had no effect on the production rate of transgenic embryos. These results indicated that genetically modified rabbit blastocysts can be efficiently produced by ICSI technique.

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Corresponding author

All correspondence to: Xiao-Rong An, State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, No. 2 Yuan-Ming-Yuan West Road, Haidian District, Beijing 100094, P.R. China. Tel:/Fax: +86 10 62733463. e-mail: xra@cau.edu.cn

References

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Bredderman, P.J., Foote, R.H. & Yassen, A.M. (1964). An improved artificial vagina for collecting rabbit semen. J. Reprod. Fertil. 30, 401–3.
Buhler, T.A., Bruyere, T., Went, D.F., Stranzinger, G. & Burki, K. (1990). Rabbit beta–casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits. Biotechnology 8, 140–3.
Camaioni, A., Russo, M.A., Odorisio, T., Gandolfi, F., Fazio, V.M. & Siracusa, G. (1992). Uptake of exogenous DNA by mammalian spermatozoa—specific localization of DNA on sperm heads. J. Reprod. Fertil. 96, 203–12.
Celebi, C., Auvray, P., Benvegnu, T., Plusquellec, D., Jegou, B. & Guillaudeux, T. (2002). Transient transmission of a transgene in mouse offspring following in vivo transfection of male germ cells. Mol. Reprod. Dev. 62, 477–82.
Chan, A.W.S., Luetjens, C.M., Dominko, T., Ramalho-Santos, J., Simerly, C.R., Hewitson, L. & Schatten, G. (2000a). Transgenic ICSI reviewed: foreign DNA transmission by intracytoplasmic sperm injection in Rhesus monkey. Mol. Reprod. Dev. 56, 325–8.
Chan, A.W.S., Luetjens, C.M., Dominko, T., Ramalho-Santos, J., Simerly, C.R., Hewitson, L. & Schatten, G. (2000b). Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol. Hum. Reprod. 6, 2633.
Chang, K., Qian, J., Jiang, M., Liu, Y., Wu, M., Chen, C.D., Lai, C.K., Lo, H.L., Hsiao, C.T., Drown, L., Bolen, J.J., Huang, H.I., Ho, P.Y., Shih, P.Y., Yao, C.W., Lin, W.J., Chen, C.H., Wu, F.Y., Lin, Y.J., Xu, J. & Wang, K. (2002). Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer. BMC Biotechnol. 2, 5.
Fan, T. & Watanabe, T. (2003). Transgenic rabbits as therapeutic protein bioreactors and human disease models. Pharmacol.Therapeut. 99, 261–82.
Farre, L., Rigau, T., Mogas, T., Garci, A.R.M, Canai, M., Gomez-Foix, A.M. & Rodri Guez-Gil, J.E. (1999). Adenovirus-mediated introduction of DNA into pig sperm and offspring. Mol. Reprod. Dev. 53, 149–58.
Hirabayashi, M., Ito, K., Sekimoto, A., Hochi, S. & Ueda, M. (2001). Production of transgenic rats using young Sprague–Dawley females treated with PMSG and hCG. Exp. Anim. 50, 365–9.
Hirabayashi, M., Kato, M., Aoto, T., Sekimoto, A., Ueda, M., Miyoshi, I., Kasai, N. & Hochi, S. (2002). Offspring derived from intracytoplasmic injection of transgenic rat sperm. Transgenic Res. 11, 221–8.
Hirabayashi, M., Kato, M., Ishikawa, A., Kaneko, R., Yagi, T. & Hochi, S. (2005). Factors affecting production of transgenic rats by ICSI-mediated DNA transfer: effects of sonication and freeze-thawing of spermatozoa, rat strains for sperm and oocyte donors, and different constructs of exogenous DNA. Mol. Reprod. Dev. 70, 422–8.
Horan, R., Powell, R., McQuaid, S., Gannon, F. & Houghton, J.A. (1991). Association of foreign DNA with porcine spermatozoa. Arch. Androl. 26, 8392.
Huguet, E. & Esponda, P. (1998). Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa. Mol. Reprod. Dev. 51, 4252.
Huguet, E. & Esponda, P. (2000). Generation of genetically modified mice by spermatozoa transfection in vivo: preliminary results. Mol. Reprod. Dev. 56, 243–7.
Kaneko, T., Moisyadi, S., Suganuma, R., Hohn, B., Yanagimachi, R. & Pelczar, P. (2005). Recombinase-mediated mouse transgenesis by intracytoplasmic sperm injection. Theriogenology 64, 1704–15.
Kato, M., Yamanouchi, K., Ikawa, M., Okabe, M., Naito, K. & Tojo, H. (1999). Efficient selection of transgenic mouse embryos using EGFP as a marker gene. Mol. Reprod. Dev. 54, 43–8.
Kato, M., Ishikawa, A., Kaneko, R., Yagi, T., Hochi, S. & Hirabayashi, M. (2004). Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study. Mol. Reprod. Dev. 69, 153–8.
Lai, L.X., Sun, Q.Y., Wu, G.M., Murphy, C.N., Kuhholzer, B., Park, K.W., Bonk, A.J., Bill, N., Day, B.N. & Prather, R.S. (2001). Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes. Zygote 9, 339–46.
Lavitrano, M. & Maione, B. (1997). The interaction of sperm cells with exogenous DNA: a role of CD4 and major histocompatibility complex class II molecules. Exp. Cell. Res. 233, 5662.
Lavitrano, M., Camaioni, A., Fazio, V.M., Dolci, S., Farace, M.G. & Spadafora, C. (1989). Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57, 717–23.
Li, Q., Hou, J., Wang, S., Guan, H., Zhang, N., Chen, Y. & An, X. (2009). Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male. Zygote 17, 157–62.
Moreira, P.N., Giraldo, P., Cozar, P., Pozueta, J., Jimenez, A., Montoliu, L. & Gutierrez-Adan, A. (2004). Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection. Biol. Reprod. 71, 1943–7.
Murakami, H., Fujimura, T., Nomura, K. & Imai, H. (2002). Factors influencing efficient production of transgenic rabbits. Theriogenology 57, 2237–45.
Nagashima, H., Fujimura, T., Takahagi, Y., Kurome, M., Wako, N., Ochiai, T., Esaki, R., Kano, K., Saito, S., Okabe, M. & Murakami, H. (2003). Development of efficient strategies for the production of genetically modified pigs. Theriogenology 59, 95106.
Ogawa, S., Hayashi, K., Jada, N., Sato, M., Kurihara, T. & Imaya, M. (1995). Gene expression in blastocysts following direct injection of DNA into testis. J. Reprod. Dev. 41, 379–82.
Osada, T., Toyoda, A., Moisyadi, S., Akutu, S., Hattori, M., Sakaki, Y. & Yanagimachi, R. (2005). Production of inbred and hybrid transgenic mice carrying large (>200 kb) foreign DNA fragments by intracytoplasmic sperm injection. Mol. Reprod. Dev. 72, 329–35.
Perry, A.C., Rothman, A., de las Heras, J.I., Feinstein, P., Monbaerts, P., Cooke, H.J. & Wakayama, T. (2001). Efficient metaphase II transgenesis with different transgene archetypes. Nat. Biotechnol. 19, 1071–3.
Perry, A.C., Wakayama, T., Kishikawa, H., Kasai, T., Okabe, M., Toyoda, Y. & Yanagimachi, R. (1999). Mammalian transgenesis by intracytoplasmic sperm injection. Science 284, 1180–3.
Sato, M., Gotoh, K. & Kimura, M. (1999). Sperm-mediated gene transfer by direct injection of foreign DNA into mouse testis. Transgenics 2, 357–69.
Sato, M., Ishikawa, A. & Kimura, M. (2002). Direct injection of foreign DNA into mouse testis as a possible in vivo gene transfer system via epididymal spermatozoa. Mol. Reprod. Dev. 69, 153–8.
Shemesh, M., Gurevich, M., Harel-Markowitz, E., Benvenisti, L., Shore, L.S. & Stram, Y. (2000). Gene integration into bovine sperm genome and its expression in transgenic offspring. Mol. Reprod. Dev. 56, 306–8.
Shen, W., Li, L., Pan, Q., Min, L., Dong, H. & Deng, J. (2006). Efficient and simple production of transgenic mice and rabbits using the new DMSO-sperm mediated exogenous DNA transfer method. Mol. Reprod. Dev. 73, 589–94.
Szczygiel, M.A., Moisyadi, S. & Ward, W.S. (2003). Expression of foreign DNA is associated with paternal chromosome degradation in intracytoplasmic sperm injection-mediated transgenesis in the mouse. Biol. Reprod. 68, 1903–10.
Venugopal, T., Anathy, V., Kirankumar, S. & Pandian, T.J. (2004). Growth enhancement and food conversion efficiency of transgenic fish Labeo rohita. J. Exp. Zool. 301A, 477–90.
Wang, H.J., Lin, A.X., Zhang, Z.C. & Chen, Y.F. (2001). Expression of porcine growth hormone gene in transgenic rabbits as reported by green fluorescent protein. Anim. Biotechnol. 12, 101–10.
Wang, H.J., Lin, A.X. & Chen, Y.F. (2003). Association of rabbit sperm cells with exogenous DNA. Anim. Biotechnol. 14, 155–65.
Yonezawa, T., Furuhata, Y., Hirabayashi, K., Suzuki, M., Takahashi, M. & Nishihara, M. (2001). Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer. Mol. Reprod. Dev. 60, 196201.

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Production of transgenic rabbit embryos through intracytoplasmic sperm injection

  • Qiuyan Li (a1), Jian Hou (a1), Sheng Wang (a1), Yongfu Chen (a1) and Xiao-Rong An (a2)...

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