Heritable and stable gene knockdown in rats

Abstract

The rat has served as an excellent model for studies on animal physiology and as a model for human diseases such as diabetes and alcoholism; however, genetic studies have been limited because of the inability to knock out genes. Our goal was to produce heritable deficiencies in specific gene function in the rat using RNA interference to knock down gene expression in vivo. Lentiviralmediated transgenesis was used to produce rats expressing a short hairpin RNA targeting Dazl, a gene expressed in germ cells and required for fertility in mice [Ruggiu, M., Speed, R., Taggart, M., McKay, S. J., Kilanowski, F., Saunders, P., Dorin, J. & Cooke, H. J. (1997) Nature 389, 73-77]. Germ-line transmission of the transgene occurred, and its expression correlated with significant reductions in DAZL protein levels and male sterility, and the knockdown was stable over multiple generations (F 1-F3). This study demonstrates an efficient system by which directed reverse genetic analysis can now be performed in the rat.Dazl ͉ fertility ͉ RNA interference T he Norway rat was the first mammal to be domesticated for scientific purposes, and over the last 150 years it has proven an invaluable research model, in part because of its size, fecundity, and ability to learn new behaviors with relative ease (1). The generation of new rat genetic models for the study of human diseases has been possible using forward genetic methods such as random mutagenesis and conventional transgenesis or through the discovery of serendipitous spontaneous mutations (2). However, reverse genetic methods crucial for analyzing the function of specific genes of interest are severely limited in the rat. Our goal was to produce heritable and stable deficiencies in specific gene function in this animal model. Gene inactivation through homologous recombination in ES cells is still not possible in the rat because of the lack of pluripotent ES cell lines similar to those available from the mouse. Instead, we chose to test an RNA interference (RNAi) approach that relies on the generation of transgenic rats.RNAi is a reverse genetic technology that allows one to down-regulate gene expression by introducing into cells a short (Ϸ19-to 21-nt) double-stranded RNA that is complementary to a target gene (3). In combination with the recent expansion of available genome sequence information, RNAi has provided a powerful tool affecting many areas of biological research. Whereas methods are now well established for performing RNAi in vitro using short interfering RNA, the application of RNAi to knock down gene expression in vivo is far less common. In most cases RNAi is achieved in vivo only transiently and is targeted to a particular tissue. Stable RNAi is usually accomplished by genetic modification of cells such that they carry a piece of DNA that contains a ubiquitous promoter (e.g., the Pol III promoters U6 or H1) that drives expression of a short hairpin RNA (shRNA). The shRNA is then processed to short interfering RNA by cellular machinery. Recent studies have shown that gen...

Spermatogonial Stem Cell Self-Renewal Requires OCT4, a Factor Downregulated During Retinoic Acid-Induced Differentiation

“…1). DAZL is a germ cell factor that is important for spermatogonial differentiation, and possibly for other stages of germ cell development, but was not expected to affect SSC colonization [44,[53][54][55][56]. From these results we conclude that OCT4 is required for SSC function.…”

“…pLLU2G contains the U6 promoter followed by an HpaI restriction site for introducing oligonucleotides coding for an shRNA, the Ubiquitin C promoter driving enhanced green fluorescent protein (eGFP) expression (Ubc-GFP), and elements for lentiviral production [44,45]. pLLU2G-Oct3 contains the following sequence in the HpaI site: GAACCGTGT-GAGGTGGAACTTCAAGAGAGTTCCACCTCACACGGTTCTT-TTTT.…”

“…pLLU2G-Oct3 can efficiently knock down both mouse and rat OCT4; the target site matches rat Oct4 mRNA and has two mismatches to the mouse Oct4 mRNA at nucleotides 18 and 19 of the target site. pLLU2G-Dazl1 is described in [44]. pLLNeo (no shRNA) and pLLNeo-Oct3 contain a PGK-Neo cassette (phosphoglycerate kinase promoter-neomycin resistance) in place of Ubc-GFP.…”

“…AF2Ј was a generous gift of Zhuoru Wu and is a lentiviral plasmid containing PGK-Neo-BGHpolyA and Ubiquitin C promoter-driven green fluorescent protein (GFP). Lentiviral particles carrying each of these vectors were produced as described [44,47]. …”

Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats

“…Therefore, they have been used for the generation of transgenic models in various species, including overexpression and genesilencing studies in mice (7)(8)(9) and rats (10)(11)(12). Temporal inactivation of genes employing inducible systems for the expression of shRNAs have been developed in vitro (13,14).…”