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Targeted Suppression of Connexin 43 in Ovine Preimplantation Embryos by RNA Interference Using Long Double-stranded RNA
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 Title & Authors
Targeted Suppression of Connexin 43 in Ovine Preimplantation Embryos by RNA Interference Using Long Double-stranded RNA
Yan, Zhen; Ma, Yu Zhen; Liu, Dong jun; Cang, Ming; Wang, Rui; Bao, Shorgan;
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RNA interference (RNAi) is an acknowledged useful and effective tool to study gene function in various cells. Here, we suppressed the Connexin 43 (Cx 43) gene expression during in vitro development of ovine pre-implantation embryos using the RNAi method. The 353 bp Cx 43 double-stranded RNA was microinjected into in vitro fertilized ovine zygotes, and the levels of target mRNA and protein were investigated. Control groups included uninjected zygotes or those injected with RNase-free water. The dsRNA injection resulted in the specific reduction of Cx 43 transcripts as analyzed by quantitative real-time RT-PCR and decreased protein levels as shown by Western blot analysis at the blastocyst stage. Microinjection of Cx 43 dsRNA led to 20.3%, 21.7% and 34.5% blastocyst rates and 19.2%, 37.5% and 41.3% hatched blastocyst rates in Cx 43 dsRNA-injected, water-injected and uninjected groups, respectively. Then the RNAi could not significantly affect cell number and cell death rates of blastocysts. Therefore, suppression of Cx 43 dsRNA and proteins did not apparently affect the development potential of ovine pre-implantation embryos but may play a role in embryo quality. RNAi technology is a promising approach to study gene function in early ovine embryogenesis.
Connexin 43;RNA Interference;Ovine Embryo;
 Cited by
Adona, P. R., P. R. Pires, M. D. Quetglas, K. R. Schwarz and C. L. Leal. 2008. Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine. Anim. Reprod. Sci. 104:389-397 crossref(new window)

Betteridge, K. J., C. Smith, R. B. Stubbings, K. P. Xu and W. A. King. 1989. Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro. J. Reprod. Fertil. Suppl. 38:87-98

Bevilacqua, A., R. Loch-Caruso and R. P. Erickson. 1989. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc. Natl. Acad. Sci. USA. 86:5444-5448 crossref(new window)

Cabot, R. A., M. Hannink and R. S. Prather. 2002. CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage. Biol. Reprod. 67:814-819 crossref(new window)

Cabot, R. A. and R. S. Prather. 2003. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol. Reprod. Dev. 64:292-301 crossref(new window)

Enright, B. P., P. Lonergan, A. Dinnyes, T. Fair, F. A. Ward, X. Yang and M. P. Boland. 2000. Culture of in vitro produced bovine zygotes in vitro vs. in vivo: implications for early embryo development and quality. Theriogenology 54:659-673 crossref(new window)

Ewart, J. L., M. F. Cohen, R. A. Meyer, G. Y. Huang, A. Wessels, R. G. Gourdie, A. J. Chin, S. M. Park, B. O. Lazatin, S. Villabon and C. W. Lo. 1997. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development 124:1281-1292

Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811 crossref(new window)

Ghassemifar, M. R., J. J. Eckert, F. D. Houghton, H. M. Picton, H. J. Leese and T. P. Fleming. 2003. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol. Hum. Reprod. 9:245-252 crossref(new window)

Hardy, K. 1997. Cell death in the mammalian blastocyst. Mol. Hum. Reprod. 3:919-925 crossref(new window)

Hu, X., S. Hipolito, R. Lynn, V. Abraham, S. Ramos and F. Wong-Staal. 2004. Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus. Nucleic Acids Res. 32:4609-4617 crossref(new window)

Larue, L., M. Ohsugi, J. Hirchenhain and R. Kemler. 1994. Ecadherin null mutant embryos fail to form a trophectoderm epithelium. Proc. Natl. Acad Sci. USA. 91:8263-8267 crossref(new window)

Lee, S., N. B. Gilula and A. E. Warner. 1987. Gap junctional communication and compaction during preimplantation stages of mouse development. Cell 51:851-860 crossref(new window)

Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408 crossref(new window)

Lonergan, P., D. Rizos, A. Gutierrez-Adan, P. M. Moreira, B. Pintado, J. de la Fuente and M. P. Boland. 2003. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol. Reprod. 69:1424-1431 crossref(new window)

Lykke-Andersen, K., M. J. Gilchrist, J. B. Grabarek, P. Das, E. Miska and M. Zernicka-Goetz. 2008. Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition. Mol. Biol. Cell 19:4383-4392 crossref(new window)

MacIntyre, D. M., H. C. Lim, K. Ryan, S. Kimmins, J. A. Small and L. A. MacLaren. 2002. Implantation-associated changes in bovine uterine expression of integrins and extracellular matrix. Biol. Reprod. 66:1430-1436 crossref(new window)

Nganvongpanit, K., H. Muller, F. Rings, M. Gilles, D. Jennen, M. Holker, E. Tholen, K. Schellander and D. Tesfaye. 2006. Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA. Mol. Reprod. Dev. 73:153-163 crossref(new window)

Paradis, F., C. Vigneault, C. Robert and M. A. Sirard. 2005. RNA interference as a tool to study gene function in bovine oocytes. Mol. Reprod. Dev. 70:111-121 crossref(new window)

Prather, R. S. and N. L. First. 1993. Cell-to-cell coupling in earlystage bovine embryos: A preliminary report. Theriogenology 39:561-567 crossref(new window)

Reaume, A. G., P. A. de Sousa, S. Kulkarni, B. L. Langille, D. Zhu, T. C. Davies, S. C. Juneja, G. M. Kidder and J. Rossant. 1995. Cardiac malformation in neonatal mice lacking connexin43. Science 267:1831-1834 crossref(new window)

Riethmacher, D., V. Brinkmann and C. Birchmeier. 1995. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc. Natl. Acad Sci. USA. 92:855-859 crossref(new window)

Rizos, D., A. Gutierrez-Adan, S. Perez-Garnelo, J. De La Fuente, M. P. Boland and P. Lonergan. 2003. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol. Reprod. 68:236-243

Rizos, D., P. Lonergan, M. P. Boland, R. Arroyo-Garcia, B. Pintado, J. de la Fuente and A. Gutierrez-Adan. 2002a. Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biol. Reprod. 66:589-595 crossref(new window)

Rizos, D., F. Ward, P. Duffy, M. P. Boland and P. Lonergan. 2002b. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol. Reprod. Dev. 61:234-248 crossref(new window)

Schmittgen, T. D., B. A. Zakrajsek, A. G. Mills, V. Gorn, M. J. Singer and M. W. Reed. 2000. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods: Anal. Biochem. 285:194-204 crossref(new window)

Stein, P., F. Zeng, H. Pan and R. M. Schultz. 2005. Absence of non-specific effects of RNA interference triggered by long 471

Svoboda, P., P. Stein, H. Hayashi and R. M. Schultz. 2000. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference: Development 127:4147-4156

Tesfaye, D., P. Lonergan, M. Hoelker, F. Rings, K. Nganvongpanit, V. Havlicek, U. Besenfelder, D. Jennen, E. Tholen and K. Schellander. 2007. Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct. Mol. Reprod. Dev. 74:978-988 crossref(new window)

Wang, S., Y. Liu, G. R. Holyoak, R. C. Evans and T. D. Bunch. 1998. A protocol for in vitro maturation and fertilization of sheep oocytes. Small Rumin. Res. 29:83-88 crossref(new window)

Wianny, F. and M. Zernicka-Goetz. 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2:70-75 crossref(new window)

Winer, J., C. K. Jung, I. Shackel and P. M. Williams. 1999. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal. Biochem. 270:41-49 crossref(new window)

Wrenzycki, C., D. Herrmann, J. W. Carnwath and H. Niemann. 1996. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J. Reprod. Fertil. 108:17-24 crossref(new window)

Wrenzycki, C., D. Herrmann, J. W. Carnwath and H. Niemann. 1999. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev. 53:8-18 crossref(new window)

Wrenzycki, C., D. Herrmann, L. Keskintepe, A. Martins, Jr., S. Sirisathien, B. Brackett and H. Niemann. 2001. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum. Reprod. 16:893-901 crossref(new window)