Gene targeting in rats using transcription activator-like effector nucleases
Introduction
The rat is considered one of the most important models in biomedical research [1]. Rat models have been shown to have many similarities to human diseases, including neurodegenerative disease, nephropathy, breast cancer and rheumatoid arthritis [2], [3], [4], [5]. Rats are approximately ten times larger than mice allowing easier collection of tissue from small structures serial blood sampling and chirurgical procedures.
Over the two decades, several technologies have been developed to modify the rat genome. These technologies include pronuclear microinjection [6], [7], lentiviral transgenesis [8], [9], [10], N-ethyl-N-nitroso urea mutagenesis [11], [12], transposon mutagenesis [13], [14], [15], embryonic stem cells [16], [17], [18], zinc-finger nuclease (ZFNs) [19], [20], [21], [22], [23] transcription activator-like effector (TALE) nucleases [24] and recently CRISPR-Cas9 system [25], [26].
TALE nucleases are hybrid molecules consist on modular repeats, each recognizing one base pair, fused of the catalytic domain of the FokI endonuclease. Two monomers of TALE nucleases recognizing contiguous sequences in both DNA strands allow dimerization of the FokI nuclease between the two TALE nucleases and DNA cleavage in this intervening sequence. During the process of DNA repair (Fig. 1) NHEJ generates deletions and insertions with a consequent rupture of the coding frame [21], [22], [27], [28], early stop codon generation and subsequent mRNA degradation [29]. In contrast, homologous recombination (HR) is a quite rare event essential for gene targeting in ES cells [27] but when a double strand DNA breaks is introduced the efficiency of HR is elevated by 3–4 logs [30]. The process of HR increased by the double strand DNA break allows the incorporation of exogenous sequences either placed between the homologous arms of a donor DNA sequence or as DNA oligonucleotides [22], [29]. This use of gene-specific nucleases coupled to the HR technique has applied to several species: plants, mouse, rats [22], [23], [24], [25], [26], [27], [28], [29], [31], [32], [33]. TALE nucleases have shown low and minimal off target activity [34]. Nevertheless, the off effects are dose dependent and at high concentrations of TALE nucleases, such as when using mRNA and not DNA encoding sequences, off effects were detected [24].
The use of TALE nuclease has been successfully employed to induce precise genome modifications in many other species: nematodes, mice, zebrafish, pigs, etc.… [35].
Two major advantages of this approach are the ability to apply ZFNs/TALE nucleases or /CRISPR/Cas9 to any rat strain and to accelerate the generation of knockout animals (∼4 months) vs. the use of embryonic stem cells (>12 months) [36]. The use of these engineered nucleases for genome editing was designated the method of the year 2011 by Nature Methods [37].
In this article, we describe a protocol which we used to generate several new knockout rats.
Section snippets
TALE nucleases
The generation of TALE nucleases is described in other manuscripts of this issue. They can be generated through access to public sources of reagents and protocols (Golden Gate (https://www.addgene.org/TALeffector/goldengateV2/), FLASH-assembly (http://talengineering.org/), http://www.jounglab.org/resources.htm as well as purchased from commercial sources.
Usually, 2–3 TALE nucleases are generated to target a given sequence and the most effective in vitro transfection of rat cells is then used in
In vitro transcription of TALE nucleases mRNA
The TALE nucleases plasmids may have a T7 promoter upstream of the TALE nucleases (Fig. 1) whereas others may have a T3 or SP6 promoter.
- 1.
Linearize 20 μg of TALE nucleases expression plasmid DNA using an enzyme situated 3′ TALE nuclease expressing sequences and 5′ of the polyA sequence (XbaI in the plasmid shown in Fig. 1) in 100 μl reaction containing 1× buffer, 1× BSA, and 80 units of enzyme restriction, at 37 °C for 2 h.
- 2.
Extract the reactions with 100 μl of phenol/chloroform, pH 8.0, and centrifuge
Acknowledgment
This work was in part funded by Region Pays de la Loire, Biogenouest, IBiSA and TEFOR (Investissements d’Avenir, French government).
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