Production of knockdown rats by lentiviral transduction of embryos with short hairpin RNA transgenes.
The primary method for determining the function of a gene in rodents has been to make a knockout mouse through homologous recombination in embryonic stem cells. However, with the advent of RNA interference (RNAi) technology, new methods for studying gene function are now possible in a wide array of animals. We describe a protocol for knocking down a gene of interest in vivo in rats by stably expressing a short hairpin RNA (shRNA). Transgenic rats are produced using a simple and efficient procedure for transducing single-cell embryos with a lentiviral vector. The vector described is designed to result in ubiquitous expression of shRNA. Thus, it is well suited to study genes expressed specifically in male germ cells in which the predicted phenotype would be male sterility. This system has been used to generate a transgenic line with stable and heritable knockdown of the gene Deleted in Azoospermia-like (Dazl), resulting in male sterility and germline transmission of the transgene through females.
Dann
Christina Tenenhaus
CT
Department of Pharmacology and Cecil H and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Garbers
David L
DL
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004624:Embryo Transfer; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D018095:Germ-Line Mutation; D007248:Infertility, Male / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D011247:Pregnancy; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics; D051381:Rats; D014161:Transduction, Genetic; D014162:Transfection
C512808:DAZL protein, rat; D017931:DNA Primers; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins