Trends in Genetics
Volume 26, Issue 12, December 2010, Pages 510-518
Journal home page for Trends in Genetics

Review
Gene targeting in the rat: advances and opportunities

https://doi.org/10.1016/j.tig.2010.08.006Get rights and content

The rat has long been a model favored by physiologists, pharmacologists and neuroscientists. However, over the past two decades, many investigators in these fields have turned to the mouse because of its gene modification technologies and extensive genomic resources. Although the genomic resources of the rat have nearly caught up, gene targeting has lagged far behind, limiting the value of the rat for many investigators. In the past two years, advances in transposon- and zinc finger nuclease (ZFN)-mediated gene knockout as well as the establishment and culturing of embryonic and inducible pluripotent stem cells have created new opportunities for rat genetic research. Here, we provide a high-level description and the potential uses of these new technologies for investigators using the rat for biomedical research.

Section snippets

Using the rat in biomedical research

The rat was the first mammalian species domesticated for scientific research, with work dating back to before 1850. Some of the first genetic studies in animals demonstrated that rat coat color is a Mendelian trait [1]. The prevalence of the rat in biomedical research is second only to humans, and there are more scientific publications using rats than any other model system based on PubMed searches. As a model system, the rat genomic toolbox is rich [2], and new sequencing technologies are

Mutagenesis via sperm manipulation

One potential access point to manipulate the rat genome is the male gamete. Investigators have made significant strides in determining the conditions for isolating, culturing and utilizing rat spermatogonial stem cells (SSCs) 6, 7, 8. Using a transgenic rat that has expressed enhanced green fluorescent protein (eGFP) exclusively in the germline, SSCs can be separated from other somatic cells and cultured for up to 12 passages 6, 7. These cells can be transfected with a selectable plasmid (a

Transposon-mediated insertional mutagenesis

The generation of mutant resources by chemical mutagenesis has been complemented by the adaptation of transposon-mediated gene-trap insertional mutagenesis using the Sleeping Beauty (SB) transposon system in rats. This strategy was developed and implemented for random saturation mutagenesis in mice 11, 12 and has now been adopted in rats 13, 14. The strategy is based on the assumption that the random insertion of a gene-trap transposon into a gene is likely to cause a null mutation by

Zinc finger nucleases

ZFNs are engineered proteins that combine the highly sequence-specific DNA binding ability of multimeric zinc finger protein domains, where individual zinc finger motifs capable of binding triplets of DNA sequence are linked together, with the nuclease activity of the restriction endonuclease FokI [18]. The plasticity of the zinc finger domain allows for the design of ZFNs that can bind specifically to a broad range of sequences 19, 20. Libraries of zinc finger motifs have been engineered with

Embryonic stem cells

In mice, targeted knockout and knockin genetic engineering is most often performed in cultured ESCs and has yielded thousands of genetically modified strains. Rat ESCs have been sought for these same purposes but have never met the three criteria that define authentic ESCs: unlimited symmetrical self-renewal in vitro, comprehensive contribution to primary chimeras and the generation of functional gametes for genome transmission. Today, the major need for rat ESCs is for knocking in genes and

Induced pluripotent stem cells

iPSCs are ESC-like cells derived from humans, mice and rats that are generated by the genetic reprogramming of differentiated cells into a ground state of pluripotency [54]. The development of useful iPSCs is principally similar to ESCs; the goal is to develop a pluripotent cell type capable of differentiating into every cell type of the adult animal and which can be potentially genetically manipulated (Box 4). However, rather than taking the precursor cell from the ICM of a blastocyst, a

Concluding remarks

There are 594 strains and many more substrains of rats currently listed in RGD. The tools outlined here are now probably amenable to site-directed mutagenesis in the majority if not all of these strains. The journey from QTL mapping to gene validation techniques [5] has evolved rapidly in the past two years. Now, not only can genes be tested in the context of QTL, but they can also be knocked out in several accepted models to generate more appropriate and valuable representations of human

Disclosure statement

The authors currently collaborate in a funded effort to knockout genes using ZFN technology and provide them as a resource to the research community (Howard Jacob 1RC2Hl101681) and Howard Jacob is the Principal Investigator of the RGD (2R01HL064541). Sigma-Aldrich Inc. and MCW Inc. have a joint license agreement that could result in MCW receiving royalties for animal sales for commercially successful strains.

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