Gene targeting in the rat: advances and opportunities

Abstract

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.

Figure I

(a) The simplest gene-trap cassette consists of a splice acceptor (SA), reporter gene, and polyadenylation signal (pA). Insertion of the gene-trap cassette into an endogenous gene disrupts the normal splicing of the gene and result in the incorporation of the reporter gene and pA into the nascent messenger RNA (mRNA) which is then translated into a fusion peptide between the endogenous gene and the reporter protein. (b) The gene-trap cassette is cloned into a transposon vector (red arrowheads). Transgenic rats harboring this gene-trap transposon are bred to transposase-transgenic rats to create doubly transgenic ‘seed’ males and the transposons are mobilized in developing sperm. These events are captured by breeding the seed male to generate G1 offspring and screening for gene trap-induced mutations (m). Putative TKO mutations can then be bred to homozygosity for phenotyping.

Figure I

ZFNs can be applied to the rat embryo to knock out genes. The engineered reagents are designed, assembled and tested in vitro, either commercially or by the user. Active ZFN pairs are introduced by pronuclear mciroinjection and transferred to pseudopregnant females which have been mated with vasectomized males. The resulting offspring are screened for mutations in the target gene and confirmed by sequencing before breeding to establish a colony for phenotyping.

Figure I

Rat ESCs are derived from the inner cell mass (ICM) of blastocyst-stage embryos and cultured under conditions which allow them to maintain their pluripotent state. Media formulas containing cocktails of small molecule inhibitors with or without LIF (described in the main text) allow the pluripotent embryonic cells to propagate. Several stages of characterization are required to validate a good ESC line including marker gene expression, pluripotency assays, and karyotyping before attempting to produce chimeric animals by injecting the donor ESCs back into a host blastocyst. Chimeras are then backcrossed to the donor strain to test for germline transmission of the ESC genome. Germline competent ESCs can be genetically manipulated to produce knockout rats, are useful for undertanding stem cell biology, and are potentially applicable to tissue engineering. ^aCombined results reported by Refs [44] and [49]. Nine cell lines generated under 2i conditions from 3 rat strains were injected into host blastocysts and two cell lines demonstrated germline competency.

Figure I

iPSCs are derived by the forced expression of specific transcription factor genes, typically by viral transduction of somatic cells such as fibroblasts. Rare cells are reprogrammed into a pluripotent state, subcloned and propagated. Once established, they are characterized like the ESCs (See Box 3).