ABSTRACT
Somatic cell nuclear transfer (SCNT) is a powerful tool for the investigation of the mechanisms of nuclear remodeling. In addition, SCNT may offer the possibility of introducing targeted mutations by homologous recombination in species for which ES cell technology is not available. The rat specific features of the oocyte have long impeded the development of SCNT. We detail here the procedures developed and optimized during the last several years for the optimization of rat cloning.
Subject(s)
Cloning, Organism/methods , Nuclear Transfer Techniques , Rats/genetics , Animals , Cells, Cultured , Embryo Transfer/methods , Embryo, Mammalian/cytology , Fibroblasts/cytology , Oocytes/cytology , Rats, Sprague-DawleyABSTRACT
In the absence of germ-line-competent ES cells in the rat, pronuclear microinjection remains an essential tool to generate transgenic rat models. However, DNA microinjection procedures first developed for mouse do not provide scientists with satisfying results when applied to rat. Here we describe optimized procedures for rat with a special focus on rat embryo production, in vitro incubation and culture, DNA pronuclear injection, and the transfer of embryos into foster females.
Subject(s)
Animals, Genetically Modified/embryology , Cell Nucleus/genetics , DNA/administration & dosage , Gene Transfer Techniques/veterinary , Microinjections/methods , Rats, Sprague-Dawley/embryology , Transgenes/physiology , Animals , Embryo Transfer , Embryo, Mammalian/cytology , Embryo, Mammalian/physiology , Female , Male , Rats , Rats, Sprague-Dawley/genetics , Zygote/physiologyABSTRACT
Because of its relevance to human physiology, the rat may provide highly predictable models for the pharmaceutical industry. Until recently, the lack of efficient tools to manipulate the rat genome has drastically limited the use of this research model. Recent advances in gene expression and transgenic systems have provided new possibilities for the generation of informative rat models. This review presents a state-of-the-art transgenic technologies in the rat and their application to biomedical research. Novel technologies enabling the faithful expression of human genes in rats are focussed on specifically.
Subject(s)
Animals, Genetically Modified , Biomedical Research/methods , Disease Models, Animal , Animals , Drug Industry , Gene Expression , Gene Targeting/methods , Humans , Rats , TransgenesABSTRACT
The laboratory rat (R. norvegicus) is a very important experimental animal in several fields of biomedical research. This review describes the various techniques that have been used to generate transgenic rats: classical DNA microinjection and more recently described techniques such as lentiviral vector-mediated DNA transfer into early embryos, sperm-mediated transgenesis, embryo cloning by nuclear transfer and germline mutagenesis. It will also cover techniques associated to transgenesis such as sperm cryopreservation, embryo freezing and determination of zygosity. The availability of several technologies allowing genetic manipulation in the rat coupled to genomic data will allow biomedical research to fully benefit from the rat as an experimental animal.