Rat Embryonic Stem Cells: Establishment and Their Use for Transgenesis

Abstract

Rats (Rattus norvegicus) have been used more extensively than mice in the research fields of neuroscience, pharmacology and toxicology. There are more than 100 rat strains with various genetic backgrounds, including some useful models for human diseases. For example, the SHR (spontaneously hypertensive rat) strain and the BB (rats spontaneously developing insulin-dependent diabetes mellitus) strain are well-established models for studying cardiovascular diseases and endocrinopathy (Okamoto, 1969; Like et al., 1982). Because of the well-understood mapping of brain functions, rats are often used for physiological studies on memory and emotion (Wood et al., 1999; van Erp et al., 2000). Furthermore, experimental studies on mammary tumors require the use of rats in which symptoms of the disease are distinct from those caused by mouse mammary tumor viruses; mouse mammary tumor viruses cause tumors in the mammary glands of mice but not those in humans or rats (Carr et al., 1981; Gould, 1986). In addition, transgenic rats have been used as model animals for human diseases (e.g., Alzheimer’s disease, autoimmunity and high-density lipoprotein [HDL] metabolism) and organ transplantation, and as animal bioreactors for protein production (Heideman, 1991; Charreau et al., 1996; Ganten, 1998). One advantage of using rats rather than mice in transgenic studies is the ease of continuous or repeated sample collection (e.g., of blood or urine) and surgery, due to their larger size, while litter size, gestation length, maturation rate, estrous cycle length, and life span of rats are all very similar to those of mice. Thus, the rat has the advantage of being a reasonably well-characterized and intermediate-sized rodent that can be maintained much more cheaply than larger animals and can often be manipulated much more easily than smaller rodents. On the other hand, the reverse genetic approach using the rats (precise and conditional gene replacements [knock-in] or loss of gene function [knock-out] at the specific locus) was considered impossible because any protocols to establish embryonic stem (ES) cell lines conventionally used in mice were not applicable to the rats. However in 2008, functional germline-competent ES cell lines have been reported (Buehr et al., 2008; Li et al., 2008). Very recently, successful production of p53 gene knock-out rats by homologous recombination in the ES cells has been achieved at last (Tong et al., 2010). The present chapter deals with an overview of attempts at producing gene-modified rats (with or without using ES cells), followed by detailed protocols for establishment of rat ES cell lines, and for successful use of the rat ES cells in transgenesis via electroporation (Hirabayashi et al., 2010a; 2010b).