Alexandre Fraichard

Transgenic Modifications of the Rat Genome

The laboratory rat (R. norvegicus) is a very important experimental animal in several fields of biomedical research. This review describes the various techniques that have been used to generate transgenic rats: classical DNA microinjection and more recently described techniques such as lentiviral vector-mediated DNA transfer into early embryos, sperm-mediated transgenesis, embryo cloning by nuclear transfer and germline mutagenesis. It will also cover techniques associated to transgenesis such as sperm cryopreservation, embryo freezing and determination of zygosity. The availability of several technologies allowing genetic manipulation in the rat coupled to genomic data will allow biomedical research to fully benefit from the rat as an experimental animal.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases

Despite the recent availability of gene‐specific nucleases, such as zinc‐finger nucleases (ZFNs) and transcription activator‐like nucleases (TALENs), there is still a need for new tools to modify the genome of different species in an efficient, rapid, and less costly manner. One aim of this study was to apply, for the first time, engineered meganucleases to mutate an endogenous gene in animal zygotes. The second aim was to target the mouse and rat recombination activating gene 1 (Rag1) to describe, for the first time, Rag1 knockout immunodeficient rats. We microinjected a plasmid encoding a meganuclease for Rag1 into the pronucleus of mouse and rat zygotes. Mutant animals were detected by PCR sequencing of the targeted sequence. A homozygous RAG1‐deficient rat line was generated and immunophenotyped. Meganucleases were efficient, because 3.4 and 0.6% of mouse and rat microinjected zygotes, respectively, generated mutated animals. RAG1‐deficient rats showed significantly decreased proportions and numbers of immature and mature T and B lymphocytes and normal NK cells vs. littermate wild‐type controls. In summary, we describe the use of engineered meganucleases to inactivate an endogenous gene with efficiencies comparable to those of ZFNs and TALENs. Moreover, we generated an immunodeficient rat line useful for studies in which there is a need for biological parameters to be analyzed in the absence of immune responses.—Ménoret, S., Fontanière, S., Jantz, D., Tesson, L., Thinard, R., Rémy, S., Usal, C., Ouisse, L.‐H., Fraichard, A., Anegon, A. Generation of Rag1‐knockout immunodeficient rats and mice using engineered meganucleases. FASEB J. 27, 703–711 (2013). www.fasebj.org

Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins.

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

Use of genetically modified rat models for translational medicine.

Because of its relevance to human physiology, the rat may provide highly predictable models for the pharmaceutical industry. Until recently, the lack of efficient tools to manipulate the rat genome has drastically limited the use of this research model. Recent advances in gene expression and transgenic systems have provided new possibilities for the generation of informative rat models. This review presents a state-of-the-art transgenic technologies in the rat and their application to biomedical research. Novel technologies enabling the faithful expression of human genes in rats are focussed on specifically.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Procedures for Somatic Cell Nuclear Transfer in the Rat

Somatic cell nuclear transfer (SCNT) is a powerful tool for the investigation of the mechanisms of nuclear remodeling. In addition, SCNT may offer the possibility of introducing targeted mutations by homologous recombination in species for which ES cell technology is not available. The rat specific features of the oocyte have long impeded the development of SCNT. We detail here the procedures developed and optimized during the last several years for the optimization of rat cloning.