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Quality of transgenic rabbit embryos with different EGFP gene constructs

Published online by Cambridge University Press:  28 May 2010

P. Chrenek ,
M. Bauer  and
A.V. Makarevich
P. Chrenek*
Affiliation:
Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky; or Slovak University of Agriculture, Nitra, Slovak Republic.
M. Bauer
Affiliation:
Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky, Slovak Republic. Department of Botany and Genetics, Constantine the Philosopher University, Nitra, Slovak Republic.
A.V. Makarevich
Affiliation:
Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky, Slovak Republic.
*
All correspondence to Peter Chrenek. Animal Production Research Centre Nitra, Hlohovecka 2, 951 41 Luzianky; or Slovak University of Agriculture, Nitra, Slovak Republic. Tel: +421 37 654 6285. Fax: +421 37 654 6189. e-mail: [email protected]
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Summary

The aim of this study was to compare the quality of rabbit transgenic embryos obtained upon microinjection of gene constructs containing different promoters and green fluorescent proteins (CMVIE–EGFP, PGK–EGFP and CMVIE–hrGFP). Developmental rate, total cell number in hatching blastocyst stage, number of apoptotic cells, diameter of embryos, transgene integration and transgenic mosaicism were investigated.

The rate of rabbit embryos microinjected with the different gene constructs developed up to morula stage was significantly lower (p < 0.05) than that of intact (non-microinjected) rabbit embryos (66–74vs. 98%). The highest efficiency of transgene integration (15%) was found when the CMVIE–EGFP (DrdI) gene construct was used, however a significantly higher transgenic mosaicism (60%) was found in rabbit embryos using this gene. The lowest cell number was counted in rabbit transgenic embryos with CMVIE–rhGFP linearized by ScaI (115.0 ± 8.20), the highest cell number (134.0 ± 35.00) was detected in rabbit transgenic embryos carrying PGK–EGFP (Not I) gene. The highest number of apoptotic cells (2.6 ± 0.33) was recorded in rabbit transgenic embryos with the integrated CMVIE–EGFP (ClaI) transgene.

Based on these results a more suitable gene marker for rabbit transgenic embryos production and selection is the CMVIE–EGFP (ClaI) gene construct. Prior to using microinjected embryos (for embryo transfer, vitrification or ESC isolation) it is necessary to pre-select microinjected embryos with evident transgenic mosaicism.

Keywords

EmbryoGFPMosaicismQualityRabbitTransgenic
Type
Research Article
Information
Zygote , Volume 19 , Issue 1 , February 2011 , pp. 85 - 90
Copyright
Copyright © Cambridge University Press 2010

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References

Chan, A.W.S., Kukolj, G., Skalka, A.M. & Bremel, R.D. (1999). Timing of DNA integration, transgenic mosaicism and pronuclear microinjection. Mol. Reprod. Dev. 52, 406–13.3.0.CO;2-P>CrossRefGoogle ScholarPubMed
Chrenek, P. & Makarevich, A. (2005). Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres. Zygote 13, 39–44.CrossRefGoogle ScholarPubMed
Chrenek, P., Vasicek, D., Makarevich, A., Jurcik, R., Suvegova, K., Bauer, M., Parkanyi, V., Rafay, J., Batorova, A. & Paleyanda, R.K. (2005). Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos. Transgenic Res. 14, 417–28.CrossRefGoogle ScholarPubMed
Duszewska, A.M., Lipinski, D., Piliszek, A., Slomski, R., Plawski, A., Wojdan, J., Gawrin, W., Juzwa, W., Zeyland, J., Wenta-Muchalska, E. & Reklewski, Z. (2004). Controversial aspect of using GFP as a marker for the production of transgenic cattle. Pol. J. Vet. Sci. 7, 241–9.Google ScholarPubMed
Fabian, D., Makarevich, A.V., Chrenek, P., Bukovská, A. & Koppel, J. (2007). Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos. Theriogenology 68, 1271–81.CrossRefGoogle ScholarPubMed
Fouladi-Nashta, A.A. (2005). Differential staining combined with TUNEL labelling to detect apoptosis in preimplantation bovine embryos. Reprod. Biomed. Online 10, 497–502.CrossRefGoogle ScholarPubMed
Gutierrez-Adan, A., Rizos, D., Fair, T., Moreira, P.N., Pintado, B., de la Fuente, J., Boland, M.P. & Lonergan, P. (2004). Effect of speed of development on mRNA expression pattern in early bovine embryos cultured in vivo or in vitro. Mol. Reprod. Dev. 68, 441–8.CrossRefGoogle ScholarPubMed
Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y. & Okabe, M. (1995). A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP). FEBS Lett. 375, 125–8.CrossRefGoogle ScholarPubMed
Jurisicova, A. & Acton, B.M. (2004). Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development. Reproduction 128, 281–91.CrossRefGoogle ScholarPubMed
Khurana, N.K. & Niemann, H. (2000). Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos. Theriogenology 54, 741–56.CrossRefGoogle ScholarPubMed
Krisher, R.L., Gibbons, J.R., Canseco, R.S., Johnson, J.L., Russell, C.G., Notter, D.R., Velander, W.H. & Gwazdauskas, F.C. (1994). Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos. Transgenic Res. 3, 226–31.CrossRefGoogle ScholarPubMed
Liu, L., Trimarchi, J.R., Keefe, D.L. (1999). Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol. Biol. Reprod. 61, 1162–9.CrossRefGoogle ScholarPubMed
Makarevich, A.V., Chrenek, P., Žilka, N., Pivko, J. & Bulla, J. (2005). Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses. Zygote 13, 1–13.Google ScholarPubMed
Makarevich, A.V., Chrenek, P. & Fl'ak, P. (2006). The influence of microinjection of foreign gene into the pronucleus of fertilized egg on the preimplantation development, cell number and diameter of rabbit embryos. Asian–Australasian J. Anim. Sci. 19, 171–5.CrossRefGoogle Scholar
Mori, M., Otoi, T. & Suzuki, T. (2002). Correlation between the cell number and diameter in bovine embryos produced in vitro. Reprod. Dom. Anim. 37, 181–4.CrossRefGoogle ScholarPubMed
O'Neill, G.T. (1995). Detection of transgenes in the preimplantation stage embryo. In Transgenic Animals. Generation and Use. Part III, Section A (ed. Houdebine, M-L.), pp. 225–31. The Netherlands: Harwood Academic Publishers.Google Scholar
Page, R.L., Bulter, S.P., Subramanian, A., Gwazdauskas, F.C., Johnson, J.L. & Velander, W.H. (1995). Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. Transgenic Res. 4, 353–60.CrossRefGoogle ScholarPubMed
Popelkova, M., Chrenek, P., Pivko, J., Makarevich, A.V., Kubovicova, E. & Kacmarik, J. (2005). Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification. Zygote 13, 283–93.Google ScholarPubMed
Rosochacki, S.J., Kozikova, L.V., Korwin-Kossakowski, M., Matejczyk, M., Poloszynowicz, J. & Duszewska, A.M. (2003). Noninvasive fluorescent screening of microinjected bovine embryos to predict transgene integration. Folia Biologica (Krakow) 51, 97–104.Google ScholarPubMed
Schwartzman, R.A. & Cidlowski, J.A. (1993). Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocrine Rev. 14, 133–51.Google ScholarPubMed
Shi, W., Dirim, F., Wolf, E., Zakhartchenko, V. & Haaf, T. (2004). Methylation reprogramming and chromosomal aneuploidy in in vivo fertilized and cloned rabbit preimplantation embryos. Biol. Reprod. 71, 340–7.CrossRefGoogle ScholarPubMed
Shu-Zhen, Liu, Jiang, M.X., Yan, L.Y., Jiang, Y., Ouyang, Y.C., Sun, Q.Y. & Chen, D. Y. (2005). Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments. Mol. Reprod. Dev. 72, 48–53.Google Scholar
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.CrossRefGoogle ScholarPubMed
Warner, C.M., Exley, G.E., McElhinny, A.S. & Tang, C. (1998). Genetic regulation of preimplantation mouse embryo survival. J. Exp. Zool. 282, 272–9.3.0.CO;2-U>CrossRefGoogle ScholarPubMed