Trends in Genetics
ReviewGene targeting in the rat: advances and opportunities
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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