Generating mutant rats using the Sleeping Beauty transposon system

Abstract

The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3′-rapid amplification of cDNA ends (3′RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.

Introduction

The laboratory rat (Rattus norvegicus) remains the major animal model system inside the pharmaceutical and biomedical research industries largely because “size does matter”. Importantly, rat models tend to be disease based, generating more information about the actual disease of interest, rather than just being an animal model. Unfortunately, geneticists have long preferred the mouse model because of its smaller size, which simplified housing requirements, and the availability of many coat color and mutants exhibiting Mendelian patterns of inheritance. In addition, the availability of mouse embryonic stem (ES) cells for gene-specific characterization and knockout studies has made this model indispensable. Although the availability of rat transgenic technologies have allowed advances in the field of gene-specific analysis, the lack of rat ES cells has hampered any further progress in this field. Germline-competent rat ES cells has been reported recently and may revolutionize this field [1], [2]. However, the robustness for large-scale generation of mutant rats remains to be tested. Chemical mutagenesis has been widely used in the rat [3], however, a large portion of induced mutations are point-mutation and thus not null alleles in most cases, and moreover, identifying the causative gene seems to be a challenging task by this method.

Sleeping Beauty (SB) transposon system is a novel genetic tool developed around a decade ago [4]. It consists of two components: The SB transposon, which is excised and reinserted into other locations of the genome, and the SB transposase catalyzes this reaction. Since most of the vertebrate transposase genes are inactivated during evolution, the SB transposase was reconstructed by extensive manipulation of inactive transposase copies from the fish genome. The SB transposon contains inverted repeat/direct repeat (IR/DR) elements at either ends (Fig. 1a), which are essential sequence for the recognition and mobilization by the SB transposase. Recently, others and we have demonstrated the effectiveness of SB for large-scale germline mutagenesis in mice [5], [6], [7]. Analysis of the SB transposition sites revealed preference of transposition on the donor-site chromosome, which varied between 60% to 80% amongst SB transposon-transgenic lines, whereas remaining 20–40% was distributed in various locations of the genome [5], [6]. Many of the transpositions on the donor-site chromosome were clustered within 3–4 Mb from the donor site [5], [6]. Others and we further applied the SB transposon system in rats, and succeeded in rapid generation of mutants [8], [9].

In the present paper, we describe a protocol for the generation and analysis of the mutant rats based on the SB transposon vectors developed in our laboratory [8]. It should be noted that various transposon systems have been developed recently and some of them have been shown to be active in mammalian cells, such as Tol2 [10], PiggyBac [11], Minos [12], and Frog Prince [13]. Although their application for insertional mutagenesis in rat remains to be addressed, it is conceivable that some of them will have different characteristics compared with SB, such as different patterns of transposition distribution [14] and different rates of transposition events. Therefore, it is highly likely that these DNA-type transposable elements will complement SB as a mutagenesis tool. Most of the protocol described in this paper can be easily modified and applicable to other transposon systems by simple modification.