Research Article: Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases

Date Published: January 25, 2010

Publisher: Public Library of Science

Author(s): Tomoji Mashimo, Akiko Takizawa, Birger Voigt, Kazuto Yoshimi, Hiroshi Hiai, Takashi Kuramoto, Tadao Serikawa, Ellen A. A. Nollen.

Abstract: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc.

Partial Text: Although several strategies are available for producing a wide variety of genomic alterations in the mouse, the same cannot be said of the rat. Rat ES cells [1], [2] and induced pluripotent stem cells (iPS) [3], [4] are available, but the culture conditions for these cells and the methodology for inducing homologous recombination are imperfect [5]. Rat spermatogonial stem cells (SSC) have also been isolated and cultivated in vitro but their yield proved unsatisfactory in terms of their ability to undergo homologous recombination [6], [7]. Besides these methods which are based on the in vitro genetic engineering of pluripotent stem cells, transposon-mediated mutagenesis [8] and N-ethyl-N-nitrosourea (ENU) mutagenesis [9], [10] have been used with some success for producing mutations in the rat genome. We recently reported on a high-throughput gene-driven strategy which uses the mutagen ENU and the Mu-transposition reaction (MuT-POWER) to rapidly detect induced mutations. This was in addition to our investigation of intracytoplasmic sperm injection (ICSI) for recovering heterozygous genotypes of interest out of a large sperm cell repository [11], [12]. However, even if a large number of mutant strains already exists or may potentially be available, targeted modification or disruption of specific DNA regions is difficult to achieve. Even in the case of our gene-driven strategy, X-linked mutations are impossible to obtain because of the breeding protocol which is used [11].

In this study, we proved that targeted gene disruption using ZFN technology works well and provides for several advantages and possibilities when used in rats. First and foremost, knockout rats can be created in a four- to six-month time frame and with high efficiency at more than 20%. This is more favorable than the ES cell-based method for mice that usually takes 12–18 months. Given the high rate of germ line transmission, preliminary phenotypic analysis can be performed on G1 animals after intercrossing the initial G0 founders, thereby saving time and effort. Second, gene-targeting with ZFNs does not seem to be strain-dependent (unpublished data) and accordingly can be performed with any inbred strain. This is of great advantage since other techniques like ENU mutagenesis differ in their efficiency when used with different strains. This provides a straight forward strategy for directly employing targeted gene disruption in the existing strain, thereby bypassing tedious and time-consuming backcrossing steps that generally take two to three years to complete. Third, ZFNs can be used to induce a wide variety of allelic changes covering small or wide deletions or insertions. They may be used to produce frameshifts or small in-frame deletions such as the 3-bp deletion that we observed. Given the reports on successful ZFN-targeted gene modification or correction by homologous recombination in mammalian cell cultures [15], [24], [25], it should be feasible to archive targeted knock-in technologies that have thus been far inaccessible without rat ES cells. Finally, since ZFN technology does not rely on using species-specific embryonic stem cell lines, it should be possible to adapt it to other mammalian species such as pigs, cattle, and monkeys, where it is possible to harvest and manipulate fertilized embryos.



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