Cloning a transgene for transgenic RNAi in mouse oocytes.
RNA interference (RNAi) is a suitable method for sequence-specific post-transcriptional gene silencing for many model systems. The protocol presented here describes the cloning of a plasmid construct for use in transgenic RNAi experiments in mouse oocytes. The protocol is intended for production of a transgene by cloning an inverted repeat (IR) into the Zp3 transgenic cassette. The procedure begins with the selection of sequences and formulation of the cloning strategy. Subsequently, the IR is cloned, inserted into the transgenic cassette, and characterized by sequencing. Finally, the transgene is released from the cassette, purified, and provided to the transgenic facility.
Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. svobodap@img.cas.cz
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D000818:Animals; D001419:Bacteria / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology*; D010957:Plasmids / Q000378:metabolism; D034622:RNA Interference; D012091:Repetitive Sequences, Nucleic Acid; D019076:Transgenes