克隆用于小鼠卵母细胞中转基因RNA干扰的转基因

Cloning a transgene for transgenic RNAi in mouse oocytes.

作者信息

Svoboda Petr

机构信息

Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.

出版信息

Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5134. doi: 10.1101/pdb.prot5134.

DOI:10.1101/pdb.prot5134

PMID:20147029

Abstract

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.

摘要

RNA干扰（RNAi）对于许多模型系统而言，是一种适用于序列特异性转录后基因沉默的方法。此处介绍的实验方案描述了用于小鼠卵母细胞转基因RNAi实验的质粒构建体的克隆。该实验方案旨在通过将反向重复序列（IR）克隆到Zp3转基因盒中来生产转基因。该过程始于序列的选择和克隆策略的制定。随后，克隆IR，将其插入转基因盒，并通过测序进行表征。最后，从盒中释放转基因，进行纯化，并提供给转基因设施。