BACKGROUND: RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. RESULTS: Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. CONCLUSIONS: We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.

Department of Epigenetic Regulations Institute of Molecular Genetics of the AS CR Videnska 1083 CZ 14220 Prague 4 Czech Republic

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Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009;10(2):126–139. doi: 10.1038/nrm2632. PubMed DOI

Svoboda P, Stein P, Hayashi H, Schultz RM. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development. 2000;127(19):4147–4156. PubMed

Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol. 2000;2(2):70–75. doi: 10.1038/35000016. PubMed DOI

Stein P. Microinjection of dsRNA into fully-grown mouse oocytes. Cold Spring Harb Protoc. 2009;2009(1):pdb prot5132. PubMed

Stein P, Svoboda P, Schultz RM. Transgenic RNAi in mouse oocytes: a simple and fast approach to study gene function. Dev Biol. 2003;256(1):187–193. doi: 10.1016/S0012-1606(02)00122-7. PubMed DOI

Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS. Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science. 2004;303(5655):238–240. doi: 10.1126/science.1090934. PubMed DOI

Svoboda P. RNA silencing in mammalian oocytes and early embryos. Curr Top Microbiol Immunol. 2008;320:225–256. full_text. PubMed

Babiarz JE, Ruby JG, Wang Y, Bartel DP, Blelloch R. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev. 2008;22(20):2773–2785. doi: 10.1101/gad.1705308. PubMed DOI PMC

Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S, Hodges E, Anger M, Sachidanandam R, Schultz RM. et al.Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature. 2008;453(7194):534–538. doi: 10.1038/nature06904. PubMed DOI PMC

Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T. et al.Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature. 2008;453(7194):539–543. doi: 10.1038/nature06908. PubMed DOI

Wang Q, Carmichael GG. Effects of length and location on the cellular response to double-stranded RNA. Microbiol Mol Biol Rev. 2004;68(3):432–452. doi: 10.1128/MMBR.68.3.432-452.2004. PubMed DOI PMC

Yang S, Tutton S, Pierce E, Yoon K. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol. 2001;21(22):7807–7816. doi: 10.1128/MCB.21.22.7807-7816.2001. PubMed DOI PMC

McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA. Gene silencing using micro-RNA designed hairpins. Rna. 2002;8(6):842–850. doi: 10.1017/S1355838202024032. PubMed DOI PMC

Chung KH, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD, Vojtek AB, Turner DL. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res. 2006;34(7):e53. doi: 10.1093/nar/gkl143. PubMed DOI PMC

Dickins RA, McJunkin K, Hernando E, Premsrirut PK, Krizhanovsky V, Burgess DJ, Kim SY, Cordon-Cardo C, Zender L, Hannon GJ. et al.Tissue-specific and reversible RNA interference in transgenic mice. Nat Genet. 2007;39(7):914–921. doi: 10.1038/ng2045. PubMed DOI PMC

Xia XG, Zhou H, Samper E, Melov S, Xu Z. Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet. 2006;2(1):e10. doi: 10.1371/journal.pgen.0020010. PubMed DOI PMC

Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell. 2002;9(6):1327–1333. doi: 10.1016/S1097-2765(02)00541-5. PubMed DOI

Meister G, Landthaler M, Dorsett Y, Tuschl T. Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. Rna. 2004;10(3):544–550. doi: 10.1261/rna.5235104. PubMed DOI PMC

Millar SE, Lader E, Liang LF, Dean J. Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity. Mol Cell Biol. 1991;11(12):6197–6204. PubMed PMC

Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 2002;16(8):948–958. doi: 10.1101/gad.981002. PubMed DOI PMC

Colledge WH, Carlton MB, Udy GB, Evans MJ. Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs. Nature. 1994;370(6484):65–68. doi: 10.1038/370065a0. PubMed DOI

Hashimoto N, Watanabe N, Furuta Y, Tamemoto H, Sagata N, Yokoyama M, Okazaki K, Nagayoshi M, Takeda N, Ikawa Y. et al.Parthenogenetic activation of oocytes in c-mos-deficient mice. Nature. 1994;370(6484):68–71. doi: 10.1038/370068a0. PubMed DOI

Kim MH, Yuan X, Okumura S, Ishikawa F. Successful inactivation of endogenous Oct-3/4 and c-mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs. Biochem Biophys Res Commun. 2002;296(5):1372–1377. doi: 10.1016/S0006-291X(02)02070-3. PubMed DOI

Stein P, Zeng F, Pan H, Schultz RM. Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes. Dev Biol. 2005;286(2):464–471. doi: 10.1016/j.ydbio.2005.08.015. PubMed DOI

Olson A, Sheth N, Lee JS, Hannon G, Sachidanandam R. RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs. Nucleic Acids Res. 2006. pp. D153–157. PubMed DOI PMC

Huesken D, Lange J, Mickanin C, Weiler J, Asselbergs F, Warner J, Meloon B, Engel S, Rosenberg A, Cohen D. et al.Design of a genome-wide siRNA library using an artificial neural network. Nat Biotechnol. 2005;23(8):995–1001. doi: 10.1038/nbt1118. PubMed DOI

Tafer H, Ameres SL, Obernosterer G, Gebeshuber CA, Schroeder R, Martinez J, Hofacker IL. The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol. 2008;26(5):578–583. doi: 10.1038/nbt1404. PubMed DOI

Paddison PJ, Cleary M, Silva JM, Chang K, Sheth N, Sachidanandam R, Hannon GJ. Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat Methods. 2004;1(2):163–167. doi: 10.1038/nmeth1104-163. PubMed DOI

Cao SF, Li D, Yuan Q, Guan X, Xu C. Spatial and temporal expression of c-mos in mouse testis during postnatal development. Asian J Androl. 2008;10(2):277–285. doi: 10.1111/j.1745-7262.2008.00324.x. PubMed DOI

Higgy NA, Zackson SL, van der Hoorn FA. Cell interactions in testis development: overexpression of c-mos in spermatocytes leads to increased germ cell proliferation. Dev Genet. 1995;16(2):190–200. doi: 10.1002/dvg.1020160211. PubMed DOI

Coumoul X, Li W, Wang RH, Deng C. Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system. Nucleic Acids Res. 2004;32(10):e85. doi: 10.1093/nar/gnh083. PubMed DOI PMC

Zhuang SZ, Li XJ, Zhang AL, Ma J, Zheng YH, Zhang FC. Transgenic RNAi-mediated reduction of LZP3 in Lagurus lagurus oocytes results in decreased fertilization ability in IVF. Mol Biol Rep. 2010;37(3):1253–1259. doi: 10.1007/s11033-009-9498-2. PubMed DOI

Svoboda P. Cloning a transgene for transgenic RNAi in mouse oocytes. Cold Spring Harb Protoc. 2009;2009(1):pdb prot5134. PubMed

Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK, Downward J, Ellenberg J, Fraser AG, Hacohen N. et al.Minimizing the risk of reporting false positives in large-scale RNAi screens. Nat Methods. 2006;3(10):777–779. doi: 10.1038/nmeth1006-777. PubMed DOI

Svoboda P. Off-targeting and other non-specific effects of RNAi experiments in mammalian cells. Curr Opin Mol Ther. 2007;9(3):248–257. PubMed

Svoboda P. Cloning and sequencing an inverted repeat. Cold Spring Harb Protoc. 2009;2009(1):pdb ip64. PubMed

Parsons BD, Schindler A, Evans DH, Foley E. A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One. 2009;4(12):e8471. doi: 10.1371/journal.pone.0008471. PubMed DOI PMC

Wang L, Ogburn CE, Ware CB, Ladiges WC, Youssoufian H, Martin GM, Oshima J. Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene. Genetics. 2000;154(1):357–362. PubMed PMC

Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil. 1989;86(2):679–688. doi: 10.1530/jrf.0.0860679. PubMed DOI

Nassenstein C, Kwong K, Taylor-Clark T, Kollarik M, Macglashan DM, Braun A, Undem BJ. Expression and function of the ion channel TRPA1 in vagal afferent nerves innervating mouse lungs. J Physiol. 2008;586(6):1595–1604. doi: 10.1113/jphysiol.2007.148379. PubMed DOI PMC

Tichopad A, Dilger M, Schwarz G, Pfaffl MW. Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res. 2003;31(20):e122. doi: 10.1093/nar/gng122. PubMed DOI PMC

Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002;30(9):e36. doi: 10.1093/nar/30.9.e36. PubMed DOI PMC

dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells