Methods xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Gene targeting in rats using transcription activator-like effector nucleases Séverine Ménoret a,b,c,⇑, Laurent Tesson a,b,c, Séverine Rémy a,b,c, Claire Usal a,b,c, Virginie Thépenier a,b,c, Reynald Thinard a,b,c, Laure-Hélène Ouisse a,b,c, Anne De Cian d, Carine Giovannangeli d, Jean-Paul Concordet d, Ignacio Anegon a,b,c a
Transgenic Rats Nantes IBiSA-Centre National de Recherche Scientifique, F44093 Nantes, France ITUN, CHU Nantes, F44000 Nantes, France INSERM UMR 1064-Center for Research in Transplantation and Immunology, France d INSERM U565, CNRS UMR7196, Museum National d’Histoire Naturelle, 43 Rue Cuvier, F75005 Paris, France b c
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: TALE nucleases Gene editing Knockout Rat model Homologous recombination Non-homologous end joining
a b s t r a c t The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models. Ó 2014 Published by Elsevier Inc.
1. 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 [2–5]. 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 [8–10], N-ethyl-N-nitroso urea mutagenesis [11–12], transposon mutagenesis [13–15], embryonic stem cells [16–18], zinc-finger nuclease (ZFNs) [19–23] 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 ⇑ Corresponding author. Address: INSERM UMR 1064-Center for Research in Transplantation and Immunology, 30 Bd Jean Monnet, F44093 Nantes, France. E-mail address: [email protected] (S. Ménoret).
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 [22–29,31–33]. 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
http://dx.doi.org/10.1016/j.ymeth.2014.02.027 1046-2023/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: S. Ménoret et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.027
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S. Ménoret et al. / Methods xxx (2014) xxx–xxx
Fig. 1. (A) TALE nuclease plasmid structure. (B) a basic diagram of TALEN function to create double strand breaks and stimulate NHEJ or HDR.
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. 2. Materials 2.1. 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 transgenesis. 2.1.1. TALE nucleases mRNA production 1. 20 lg of each plasmid encoding the monomers of TALE nucleases. 2. MessageMax T7 ARCA-Capped Message Transcription Kit (Epicentre Biotechnologies, Madison, WI) or equivalent. 3. A-Plus Poly (A) Polymerase tailing kit (Epicentre Biotechnologies) or equivalent. 4. MegaClear kit (Ambion, Austin, TX) or equivalent. 5. Formamide loading buffer (0.05% xylene cyanol, 0.05% bromophenol blue in formamide). 6. 1X TBE buffer (90 mM Tris base, 90 mM boric acid, 2 mM EDTA pH 8.0). 7. Agarose. 8. Ethidium bromide. 2.1.2. Screening of TALE nucleases and gene targeting in rat cells 1. C6 cells (ATCC; cat No. CCL-107). 2. F-12 medium supplemented with 5% fetal bovine serum and 15% horse serum, 3. Trypsin–EDTA (0.25%), 1X phosphate buffered saline (PBS) (Invitrogen, Carlsbad, CA).
4. Amaxa nucleofection kit V (Lonza, Cologne, Germany). 5. Masterpure DNA Preparation kit (Epicentre Biotechnologies). 2.2. Gene targeting in rats All experiments were compliant with Animal Protection Law of the French Republic (article R214-89) which is in compliance with the European Community Council recommendations for the use of laboratory animals 86/609/ECC and EU directive 2010/63/EU for animal experiments, and experiments were approved by the CEEA Pays de la Loire committee (ref CEEA-2011-45). 2.2.1. Superovulation and fertilized embryo collection 1. Immature Sprague–Dawley females (4–5 weeks old and 75–100 g in weight). (Charles River). 2. Fertile Sprague–Dawley males (2 months old, Charles River) for mating with the immature females. They should be replaced every 8–12 months. 3. Pregnant Mare’s Serum Gonadotrophin (PMSG; Intervet Laboratories Ltd., Cambridge, UK). Working solution: 125 IU/ml made up with 0.9% (w/v) NaCl. Store frozen at 20 °C in 1 ml aliquots. 4. Human chorionic gonadotrophin (hCG; Chorulon, Intervet). Working solution: 150 IU/ml made up with 0.9% (w/v) NaCl. Store at 4 °C (up to 2 weeks) in 1 ml aliquots. 5. Hyaluronidase (Sigma; cat. No. H4272). Stock solution: 10 mg/ml in M16 medium (Sigma; cat. No. M7292). Store at 20 °C (stable for several months) in 50 ll aliquots. 6. Embryo culture medium: M16 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. 7. Embryo-tested mineral oil (Sigma; cat. No. M8410). 8. 35-mm Petri dishes. 9. Egg transfer pipette (Pasteur Pipette) assembled into a mouth-operated system made up of a mouthpiece, rubber tube (40 cm) and a pipette holder. 10. Stereomicroscope with under-stage illumination. 11. Humidified incubator at 37 °C and 5% CO2. 2.2.2. Microinjection of one-cell embryos 1. Inverted microscope equipped with a 10 lens for lowmagnification work and a 40 objective for microinjection.
Please cite this article in press as: S. Ménoret et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.027
S. Ménoret et al. / Methods xxx (2014) xxx–xxx
2. Two micromanipulators for both holding and microinjection pipettes (Narishige, London, UK). 3. Microinjector (Narishige). 4. Micropipette puller (PN-30, Narishige). 5. Holding pipette (Glass capillary, Narishige; cat. No. G-1). 6. Microinjection pipette (Glass capillary with filament, Narishige; cat. No. GD-1). 7. Tips for loading capillaries with mRNAs solution (Microloader, Eppendorf). 8. Injection chamber. 2.2.3. Transfer of microinjected embryos into recipient females 1. Recipient Sprague–Dawley females (8–16 weeks of age) that have successfully had at least one litter (virgins often eat pups) (Charles River). 2. Vasectomized Sprague–Dawley males (Charles River) are needed to engender pseudopregnancy in the recipient females. Since the floors of their cages are of wire mesh veterinary rules oblige to replace them every 1 year. 3. Surgical microscope and fiber optic illumination. 4. Adrenalin (1 mg/ml). 5. Embryo transfer pipette assembled into a mouthpiece. 2.3. Analysis of gene targeting in the 10-day-old rats 2.3.1. Preparation of DNA from tail pups biopsies 1. Tissue digestion buffer: 100 mM Tris–HCl pH 8.5, 10 mM EDTA, 0.2% (w/v) SDS, 200 mM NaCl, 200 lg/ml Proteinase K. Add Proteinase K immediately before use from a freshly-made stock solution. 2. Isopropanol. 3. 70% ethanol. 4. TE: 10 mM Tris–HCl pH 8.5, 1 mM EDTA. 2.3.2. Genotyping of -modified rats by PCR and sequencing 1. Topo-TA cloning kit (Invitrogen). 2. Sequencing primers specific of the targeted gene. 3. High-fidelity polymerase (Herculase II fusion polymerase; Stratagene, La Jolla, CA; USA). 4. NE Buffer 2 (New England Biolabs, Ipswich, MA, USA). 5. T7 endonuclease I (M0302L; New England Biolabs). 6. Tissue DNA kit EZNA (Omega bio-tek). 3. Methods 3.1. Preparation of TALE nucleases mRNA 3.1.1. 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 lg of TALE nucleases expression plasmid DNA using an enzyme situated 30 TALE nuclease expressing sequences and 50 of the polyA sequence (XbaI in the plasmid shown in Fig. 1) in 100 ll reaction containing 1 buffer, 1 BSA, and 80 units of enzyme restriction, at 37 °C for 2 h. 2. Extract the reactions with 100 ll of phenol/chloroform, pH 8.0, and centrifuge at 20,000g for 10 min. 3. Transfer the aqueous phase to a clean tube and precipitate with 10 ll 3 M NaOAc and 250 ll 100% ethanol and centrifuge at top speed for 25 min at room temperature.
3
4. Decant supernatant and wash pellet with 300 ll 70% ethanol and centrifugation at top speed for 3 min. 5. Air dry the pellet for 5 min and resuspend in 20 lL of 0.1 TE. 3.1.2. Capped in vitro transcription Use an in vitro transcription kit which utilizes the T7 (or T3) RNA polymerase and incorporates a 7-methylguanosine cap. Ambion’s MessageMax T7 ARCA-capped message kit uses the anti-reverse cap analog (ARCA) and improves the yield of capped mRNA. 3.1.3. Polyadenosine-tailing reaction Immediately following in vitro transcription, use a polyadenosine (polyA)-tailing kit such as the A-plus Poly (A) Polymerase tailing kit. We have never used TALE nucleases mRNA without polyadenylation but this has been done for microinjection in livestock [30]. 3.1.4. Purification and gel visualization of mRNA 1. Immediately following the polyA-tailing reaction, use a kit such as the Ambion MegaClear kit to purify the mRNA and elute in an RNAse-free solution. 2. Measure RNA concentration and OD260/280 ratio (Our normal yield is about 30 lg/reaction, using Epicentre’s transcription and tailing kits and Ambion’s purification kit. OD260/280 is always above 2). 3. Mix 1 ll of the eluted TALE nucleases mRNA and 1 ll of formamide loading buffer (0.05% xylene cyanol, 0.05% bromophenol blue in formamide). 4. Heat to 70 °C for 3 min and place on ice. 5. Load to 1% TBE agarose gel and run in 1 TBE buffer at 12 V/cm for 20 min. New running buffer and high voltage and thus a short running time are critical for maintaining mRNA integrity during electrophoresis) with a proper size marker. TALE nucleases mRNAs runs at 1 kb as one defined band with minor smearing below the major band. 6. Dilute mRNA to 10 or 5 ng/ll final concentrations of the combination of each TALE nuclease in the mixture and store at 80 °C until use. (Cf Fig. 2:schema of mRNA preparation of TALE nucleases). 3.2. Validating TALE nucleases activity assay in cultured rat cells 1. Two transfections should be performed: one with the TALE nucleases plasmids (each containing a monomer), the other with a plasmid donor containing an eGFP expression cassette to estimate transfection efficiency and serve as a negative control for TALE nucleases activity. Transfect 2 million C6 cells with 5 lg each TALE nuclease plasmid. We used the Amaxa nucleofection kit V. 2. Two days post-transfection, harvest cells via trypsinization. 3. Chromosomal DNA is prepared with Tissue DNA kit EZNA or similar, follow manufacturer’s instructions. 4. Analyze the chromosomal DNA for mutations induced by TALE nucleases with T7 endonuclease I assay [38]. 5. PCRs were performed with a high-fidelity polymerase (Herculase II fusion polymerase) run on a gel to verify size and specific amplification (without nonspecific products). The PCR products (150 ng; 5 of 25 ll) were denatured and reannealed according to the following thermocycler conditions: 95 °C for 5 min, 95– 85 °C at 2 °C/s, 85–25 °C at 0.1 °C/s, and hold at 4 °C. Added 1 ll of NEBuffer 2, 0.5 ll (5 U) of T7 endonuclease I (M0302L), and 3.5 ll of H2O and incubated the mixture at 37 °C for 30 min. 6. Perform an electrophoresis with the mixture on a 10% polyacrylamide gel and 0.5 lg/ml. Ethidium bromide staining at 10–15 V/cm or freeze at 20° C for later analysis.
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Fig. 2. Schematic diagram for mRNA preparation. After purification of DNAs, mMESSAGE mMACHINEÒT7 Ultra kit is used to cap DNAs with ARCA and transcript them by T7 RNA polymerase with addition of poly(A) tail to make mRNA. Then, mRNAs are cleaned-up with MEGAclear™ Kit and checked on Nanodrop spectrophotometer (Agilent) to verify quantity and quality of the mRNAs.
7. Visualize gel for evidence of nuclease cleavage, which will be evident as new, shorter bands not present in the control lane. 3.3. Gene targeting in rats embryos 3.3.1. Superovulation and fertilized embryo collection Sprague–Dawley female rats are administrated gonadotropins prior to mating to increase the number of released eggs (superovulation). 1. Inject prepubescents Sprague–Dawley females rats intraperitonally with 25 IU of PMSG between 12 am and 1 pm on day -2, followed by 30 IU of hCG between 3 and 4 pm on day 0. 2. Individually mate each treated female with one fertile male overnight. On the morning of day 1, check the females for copulation plugs. 3. Sacrifice female rats by anesthesia on isofluorane steady cervical dislocation around 10 a.m. on the morning of day 1. 4. Excise the oviducts and transfer them to a dish containing PBS or M2 medium at room temperature. Embryos, enclosed by cumulus mass cells, can be released from the swollen ampullae (the upper portion of the oviduct) by gently tugging and opening the walls of the ampullae with fine forceps. 5. Transfer the embryos using an egg transfer pipette to a dish containing a pre-warmed hyaluronidase solution (500 lg/ml in PBS), which enzymatically digests the cumulus cells thus releasing the embryos. A few minutes of treatment is sufficient (longer incubation can be toxic for embryos); gentle up and down pipeting can facilitate the process. 6. Transfer the embryos to PBS to wash off the hyaluronidase solution and preserve their viability. 7. Finally, transfer the embryos in fresh pre-warned M16 embryo culture medium in a humidified 37 °C incubator under 5% CO2 until microinjection. 3.3.2. Delivery of TALE nucleases mRNA to one-cell embryos The TALE nucleases mRNAs are microinjected into the cytoplasm (mRNA) of single-cell embryos.
1. Thaw a newly frozen aliquot (10 ll) of mRNA solution at room temperature and centrifuge at 1455g for 20 s. Kept the solution on ice during the microinjection and discard after one-day session of microinjection. 2. Load approximately 2–3 ll of solution into the microinjection pipette with a microloader tip. 3. The loaded micropipette is held in place onto a micromanipulator connected to the N2 gas-operated pressure injector. 4. Transfer one-cell fertilized embryos (in batches of 30–40) in a microdrop of embryo culture medium in the injection chamber and cover with mineral oil to prevent evaporation and maintain osmolarity. 5. Mount the chamber on the stage of an inverted microscope, and monitor the injection procedure under 400 magnification. 6. Hold fertilized embryos (pronuclei visible) in place against the holding pipette using gentle negative pressure. Hold the micropipette loaded in place onto a micromanipulator. 7. Using the micromanipulator to guide the pipette, push the tip through the zona pellucida into the cytoplasm. 8. Using gentle positive pressure, the solution flows continuously from the pipette. The delivery into the cytoplasm is successful when the injected solution spreads to form a drop having a diameter approximately similar to the pronuclei’s one. 9. The microinjection pipette should be changed every 30–40 embryos. 10. After the injection, transfer the surviving embryos in embryo culture medium equilibrated at 37 °C, 5% CO2, and then keep them in a 37 °C humidified incubator under 5% CO2 until implantation. 3.3.3. Transfer of embryos into recipient females 1. Obtain pseudopregnant Sprague–Dawley females needed to host the microinjected embryos by mating sexually mature Sprague–Dawley females with vasectomized mature males, the night before the day of implantation. Confirm mating the
Please cite this article in press as: S. Ménoret et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.027
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next morning by checking for a plug. We mate 16 vasectomized males individually with females and usually obtain between 1–3 mattings. The non-mated females are used for another mating 10 days after the first attempt. 2. Transfer injected embryos into the oviduct of host females by using an embryo transfer pipette, preferably the same day as the microinjection to increase the rate of implantation. In general, no more than 24 embryos are transferred bilaterally into the oviduct. 3.4. Analysis of DNA mutations from rat tissues (Fig 3) 3.4.1. Analysis of DNA from rat tissues from 10-days-old rat 1. Harvest short tail biopsies (6 mm) 10-days-old rat. 2. Add 500 ll tissue digestion buffer to the tail biopsies in a 1.5 ml tube. 3. Incubate at 56 °C for overnight. 4. Centrifuge 18,000g at for 1 min. 5. Transfer lysate to a new microcentrifuge tube. 6. Add 500 ll isopropanol. 7. Invert tubes 20–30 times until nucleic acids precipitate. 8. Centrifuge at 18,000g for 10 min. 9. Wash nucleic acid pellet with 1 mL 70% ethanol. 10. Allow pellet to dry for 5 min. Do not dry the pellet to completion. 11. Dissolve the pellet in 100 ll TE for >15 min. 3.4.2. Analysis of gene modifications by PCR (Fig.3) 1. Design oligonucleotides that will amplify PCR products comprised between 300 and 800 bp of the region of interest from rat genome. 2. Optimize PCR conditions so that only one specific product is obtained using high-fidelity polymerase. PCR amplify the region of interest from the crude nucleic acid preparation. 3. Electrophorese the sample on a 10% polyacrylamide gel at 10– 15 V/cm or freeze at 20 °C for later analysis. 4. If the deletions are large, the visualization of PCR products on gel may allow identifying some mutated founders but all animals should be analyzed using the T7 nuclease assay and sequencing. 3.4.3. Enzymatic mismatch cleavage with T7 endonuclease I assay and sequencing 1. Perform enzymatic mismatch cleavage with T7 endonuclease I assay as described in point 3.2.
Fig. 3. T7 endonuclease I assay of rat DNA. Tails’ biopsies DNA from 4 different rats after microinjection of the same TALE nuclease were analyzed using the T7 endonuclease I assay. Rat N° 2 shows smaller bands after T7 endonuclease I treatment, indicative of a mutation.
2. The products are separated by electrophoresis to visualize evidence of T7 nuclease cleavage. The presence of mismatches is visualized by the generation of lower molecular weight bands (Fig. 3).
3.4.4. Confirmation of mutations by sequencing of PCR products 1. PCR products could be direct sequenced or cloned via TA cloning (i.e. TOPO PCR cloning system (follow the kit manufacturer’s instructions) and the presence of mutations is visualized by comparison with wild-type sequences. 2. If more than one mutation is present in the same founder animal, PCR products should be cloned in a sequencing plasmid and 10–20 bacteria colonies sequenced independently.
3.5. Results The results obtained with 4 TALE nucleases for 4 different gene targets are depicted in Table 1. We first generated rats with mutations in the IgM locus microinjecting the TALE nucleases in DNA or mRNA forms at different concentrations [22] as we had previously done for using ZFNs [19]. At the highest concentration used (10 ng/ll) we observed a toxic effect on survival of zygotes after microinjection and more obviously in birth rates and most importantly in overall mutation efficiency (mutated newborns/microinjected embryos since the number of mutated newborns was low. A key point is that the mutation rate was higher with TALE nucleases mRNA vs. DNA (4.02–10.1% vs. 0–1.81%, respectively).
Table 1 Analysis of mutations by TALE nucleases after microinjection in rat embryos. Target/construct (strain)
Route/dose (ng/ ll)
Surviving embryo rate
Birth rate⁄
Control plasmid DNA (SD) IgM/DNA (SD)
PNI/2
72.5
14.5
1.42
PNI/10 PNI/2 PNI/0.4 IC/10 IC/4 IC/0.8 IC/10 IC/2 IC/0.4 IC/5 IC/5
59 64 73 72 73 82 62.5 77 74 83 79.2
7.83 22.5 3.17 9.9 14.2 21.4 3.98 14.03 14.6 22.7 20.1
1.81 1.69 0 6.9 10.1 4.02 3.4 5.7 2.28 2.84 11.1
IgM/mRNA (SD)
X/mRNA (SD)
Y/mRNA (SD) Z/mRNA(SD)
Mutation rate⁄
% biallellic mutations
% Ins/ % Del
Size Ins/Del (%
Contents lists available at ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Gene targeting in rats using transcription activator-like effector nucleases Séverine Ménoret a,b,c,⇑, Laurent Tesson a,b,c, Séverine Rémy a,b,c, Claire Usal a,b,c, Virginie Thépenier a,b,c, Reynald Thinard a,b,c, Laure-Hélène Ouisse a,b,c, Anne De Cian d, Carine Giovannangeli d, Jean-Paul Concordet d, Ignacio Anegon a,b,c a
Transgenic Rats Nantes IBiSA-Centre National de Recherche Scientifique, F44093 Nantes, France ITUN, CHU Nantes, F44000 Nantes, France INSERM UMR 1064-Center for Research in Transplantation and Immunology, France d INSERM U565, CNRS UMR7196, Museum National d’Histoire Naturelle, 43 Rue Cuvier, F75005 Paris, France b c
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: TALE nucleases Gene editing Knockout Rat model Homologous recombination Non-homologous end joining
a b s t r a c t The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models. Ó 2014 Published by Elsevier Inc.
1. 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 [2–5]. 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 [8–10], N-ethyl-N-nitroso urea mutagenesis [11–12], transposon mutagenesis [13–15], embryonic stem cells [16–18], zinc-finger nuclease (ZFNs) [19–23] 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 ⇑ Corresponding author. Address: INSERM UMR 1064-Center for Research in Transplantation and Immunology, 30 Bd Jean Monnet, F44093 Nantes, France. E-mail address: [email protected] (S. Ménoret).
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 [22–29,31–33]. 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
http://dx.doi.org/10.1016/j.ymeth.2014.02.027 1046-2023/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: S. Ménoret et al., Methods (2014), http://dx.doi.org/10.1016/j.ymeth.2014.02.027
2
S. Ménoret et al. / Methods xxx (2014) xxx–xxx
Fig. 1. (A) TALE nuclease plasmid structure. (B) a basic diagram of TALEN function to create double strand breaks and stimulate NHEJ or HDR.
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. 2. Materials 2.1. 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 transgenesis. 2.1.1. TALE nucleases mRNA production 1. 20 lg of each plasmid encoding the monomers of TALE nucleases. 2. MessageMax T7 ARCA-Capped Message Transcription Kit (Epicentre Biotechnologies, Madison, WI) or equivalent. 3. A-Plus Poly (A) Polymerase tailing kit (Epicentre Biotechnologies) or equivalent. 4. MegaClear kit (Ambion, Austin, TX) or equivalent. 5. Formamide loading buffer (0.05% xylene cyanol, 0.05% bromophenol blue in formamide). 6. 1X TBE buffer (90 mM Tris base, 90 mM boric acid, 2 mM EDTA pH 8.0). 7. Agarose. 8. Ethidium bromide. 2.1.2. Screening of TALE nucleases and gene targeting in rat cells 1. C6 cells (ATCC; cat No. CCL-107). 2. F-12 medium supplemented with 5% fetal bovine serum and 15% horse serum, 3. Trypsin–EDTA (0.25%), 1X phosphate buffered saline (PBS) (Invitrogen, Carlsbad, CA).
4. Amaxa nucleofection kit V (Lonza, Cologne, Germany). 5. Masterpure DNA Preparation kit (Epicentre Biotechnologies). 2.2. Gene targeting in rats All experiments were compliant with Animal Protection Law of the French Republic (article R214-89) which is in compliance with the European Community Council recommendations for the use of laboratory animals 86/609/ECC and EU directive 2010/63/EU for animal experiments, and experiments were approved by the CEEA Pays de la Loire committee (ref CEEA-2011-45). 2.2.1. Superovulation and fertilized embryo collection 1. Immature Sprague–Dawley females (4–5 weeks old and 75–100 g in weight). (Charles River). 2. Fertile Sprague–Dawley males (2 months old, Charles River) for mating with the immature females. They should be replaced every 8–12 months. 3. Pregnant Mare’s Serum Gonadotrophin (PMSG; Intervet Laboratories Ltd., Cambridge, UK). Working solution: 125 IU/ml made up with 0.9% (w/v) NaCl. Store frozen at 20 °C in 1 ml aliquots. 4. Human chorionic gonadotrophin (hCG; Chorulon, Intervet). Working solution: 150 IU/ml made up with 0.9% (w/v) NaCl. Store at 4 °C (up to 2 weeks) in 1 ml aliquots. 5. Hyaluronidase (Sigma; cat. No. H4272). Stock solution: 10 mg/ml in M16 medium (Sigma; cat. No. M7292). Store at 20 °C (stable for several months) in 50 ll aliquots. 6. Embryo culture medium: M16 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. 7. Embryo-tested mineral oil (Sigma; cat. No. M8410). 8. 35-mm Petri dishes. 9. Egg transfer pipette (Pasteur Pipette) assembled into a mouth-operated system made up of a mouthpiece, rubber tube (40 cm) and a pipette holder. 10. Stereomicroscope with under-stage illumination. 11. Humidified incubator at 37 °C and 5% CO2. 2.2.2. Microinjection of one-cell embryos 1. Inverted microscope equipped with a 10 lens for lowmagnification work and a 40 objective for microinjection.
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2. Two micromanipulators for both holding and microinjection pipettes (Narishige, London, UK). 3. Microinjector (Narishige). 4. Micropipette puller (PN-30, Narishige). 5. Holding pipette (Glass capillary, Narishige; cat. No. G-1). 6. Microinjection pipette (Glass capillary with filament, Narishige; cat. No. GD-1). 7. Tips for loading capillaries with mRNAs solution (Microloader, Eppendorf). 8. Injection chamber. 2.2.3. Transfer of microinjected embryos into recipient females 1. Recipient Sprague–Dawley females (8–16 weeks of age) that have successfully had at least one litter (virgins often eat pups) (Charles River). 2. Vasectomized Sprague–Dawley males (Charles River) are needed to engender pseudopregnancy in the recipient females. Since the floors of their cages are of wire mesh veterinary rules oblige to replace them every 1 year. 3. Surgical microscope and fiber optic illumination. 4. Adrenalin (1 mg/ml). 5. Embryo transfer pipette assembled into a mouthpiece. 2.3. Analysis of gene targeting in the 10-day-old rats 2.3.1. Preparation of DNA from tail pups biopsies 1. Tissue digestion buffer: 100 mM Tris–HCl pH 8.5, 10 mM EDTA, 0.2% (w/v) SDS, 200 mM NaCl, 200 lg/ml Proteinase K. Add Proteinase K immediately before use from a freshly-made stock solution. 2. Isopropanol. 3. 70% ethanol. 4. TE: 10 mM Tris–HCl pH 8.5, 1 mM EDTA. 2.3.2. Genotyping of -modified rats by PCR and sequencing 1. Topo-TA cloning kit (Invitrogen). 2. Sequencing primers specific of the targeted gene. 3. High-fidelity polymerase (Herculase II fusion polymerase; Stratagene, La Jolla, CA; USA). 4. NE Buffer 2 (New England Biolabs, Ipswich, MA, USA). 5. T7 endonuclease I (M0302L; New England Biolabs). 6. Tissue DNA kit EZNA (Omega bio-tek). 3. Methods 3.1. Preparation of TALE nucleases mRNA 3.1.1. 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 lg of TALE nucleases expression plasmid DNA using an enzyme situated 30 TALE nuclease expressing sequences and 50 of the polyA sequence (XbaI in the plasmid shown in Fig. 1) in 100 ll reaction containing 1 buffer, 1 BSA, and 80 units of enzyme restriction, at 37 °C for 2 h. 2. Extract the reactions with 100 ll of phenol/chloroform, pH 8.0, and centrifuge at 20,000g for 10 min. 3. Transfer the aqueous phase to a clean tube and precipitate with 10 ll 3 M NaOAc and 250 ll 100% ethanol and centrifuge at top speed for 25 min at room temperature.
3
4. Decant supernatant and wash pellet with 300 ll 70% ethanol and centrifugation at top speed for 3 min. 5. Air dry the pellet for 5 min and resuspend in 20 lL of 0.1 TE. 3.1.2. Capped in vitro transcription Use an in vitro transcription kit which utilizes the T7 (or T3) RNA polymerase and incorporates a 7-methylguanosine cap. Ambion’s MessageMax T7 ARCA-capped message kit uses the anti-reverse cap analog (ARCA) and improves the yield of capped mRNA. 3.1.3. Polyadenosine-tailing reaction Immediately following in vitro transcription, use a polyadenosine (polyA)-tailing kit such as the A-plus Poly (A) Polymerase tailing kit. We have never used TALE nucleases mRNA without polyadenylation but this has been done for microinjection in livestock [30]. 3.1.4. Purification and gel visualization of mRNA 1. Immediately following the polyA-tailing reaction, use a kit such as the Ambion MegaClear kit to purify the mRNA and elute in an RNAse-free solution. 2. Measure RNA concentration and OD260/280 ratio (Our normal yield is about 30 lg/reaction, using Epicentre’s transcription and tailing kits and Ambion’s purification kit. OD260/280 is always above 2). 3. Mix 1 ll of the eluted TALE nucleases mRNA and 1 ll of formamide loading buffer (0.05% xylene cyanol, 0.05% bromophenol blue in formamide). 4. Heat to 70 °C for 3 min and place on ice. 5. Load to 1% TBE agarose gel and run in 1 TBE buffer at 12 V/cm for 20 min. New running buffer and high voltage and thus a short running time are critical for maintaining mRNA integrity during electrophoresis) with a proper size marker. TALE nucleases mRNAs runs at 1 kb as one defined band with minor smearing below the major band. 6. Dilute mRNA to 10 or 5 ng/ll final concentrations of the combination of each TALE nuclease in the mixture and store at 80 °C until use. (Cf Fig. 2:schema of mRNA preparation of TALE nucleases). 3.2. Validating TALE nucleases activity assay in cultured rat cells 1. Two transfections should be performed: one with the TALE nucleases plasmids (each containing a monomer), the other with a plasmid donor containing an eGFP expression cassette to estimate transfection efficiency and serve as a negative control for TALE nucleases activity. Transfect 2 million C6 cells with 5 lg each TALE nuclease plasmid. We used the Amaxa nucleofection kit V. 2. Two days post-transfection, harvest cells via trypsinization. 3. Chromosomal DNA is prepared with Tissue DNA kit EZNA or similar, follow manufacturer’s instructions. 4. Analyze the chromosomal DNA for mutations induced by TALE nucleases with T7 endonuclease I assay [38]. 5. PCRs were performed with a high-fidelity polymerase (Herculase II fusion polymerase) run on a gel to verify size and specific amplification (without nonspecific products). The PCR products (150 ng; 5 of 25 ll) were denatured and reannealed according to the following thermocycler conditions: 95 °C for 5 min, 95– 85 °C at 2 °C/s, 85–25 °C at 0.1 °C/s, and hold at 4 °C. Added 1 ll of NEBuffer 2, 0.5 ll (5 U) of T7 endonuclease I (M0302L), and 3.5 ll of H2O and incubated the mixture at 37 °C for 30 min. 6. Perform an electrophoresis with the mixture on a 10% polyacrylamide gel and 0.5 lg/ml. Ethidium bromide staining at 10–15 V/cm or freeze at 20° C for later analysis.
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Fig. 2. Schematic diagram for mRNA preparation. After purification of DNAs, mMESSAGE mMACHINEÒT7 Ultra kit is used to cap DNAs with ARCA and transcript them by T7 RNA polymerase with addition of poly(A) tail to make mRNA. Then, mRNAs are cleaned-up with MEGAclear™ Kit and checked on Nanodrop spectrophotometer (Agilent) to verify quantity and quality of the mRNAs.
7. Visualize gel for evidence of nuclease cleavage, which will be evident as new, shorter bands not present in the control lane. 3.3. Gene targeting in rats embryos 3.3.1. Superovulation and fertilized embryo collection Sprague–Dawley female rats are administrated gonadotropins prior to mating to increase the number of released eggs (superovulation). 1. Inject prepubescents Sprague–Dawley females rats intraperitonally with 25 IU of PMSG between 12 am and 1 pm on day -2, followed by 30 IU of hCG between 3 and 4 pm on day 0. 2. Individually mate each treated female with one fertile male overnight. On the morning of day 1, check the females for copulation plugs. 3. Sacrifice female rats by anesthesia on isofluorane steady cervical dislocation around 10 a.m. on the morning of day 1. 4. Excise the oviducts and transfer them to a dish containing PBS or M2 medium at room temperature. Embryos, enclosed by cumulus mass cells, can be released from the swollen ampullae (the upper portion of the oviduct) by gently tugging and opening the walls of the ampullae with fine forceps. 5. Transfer the embryos using an egg transfer pipette to a dish containing a pre-warmed hyaluronidase solution (500 lg/ml in PBS), which enzymatically digests the cumulus cells thus releasing the embryos. A few minutes of treatment is sufficient (longer incubation can be toxic for embryos); gentle up and down pipeting can facilitate the process. 6. Transfer the embryos to PBS to wash off the hyaluronidase solution and preserve their viability. 7. Finally, transfer the embryos in fresh pre-warned M16 embryo culture medium in a humidified 37 °C incubator under 5% CO2 until microinjection. 3.3.2. Delivery of TALE nucleases mRNA to one-cell embryos The TALE nucleases mRNAs are microinjected into the cytoplasm (mRNA) of single-cell embryos.
1. Thaw a newly frozen aliquot (10 ll) of mRNA solution at room temperature and centrifuge at 1455g for 20 s. Kept the solution on ice during the microinjection and discard after one-day session of microinjection. 2. Load approximately 2–3 ll of solution into the microinjection pipette with a microloader tip. 3. The loaded micropipette is held in place onto a micromanipulator connected to the N2 gas-operated pressure injector. 4. Transfer one-cell fertilized embryos (in batches of 30–40) in a microdrop of embryo culture medium in the injection chamber and cover with mineral oil to prevent evaporation and maintain osmolarity. 5. Mount the chamber on the stage of an inverted microscope, and monitor the injection procedure under 400 magnification. 6. Hold fertilized embryos (pronuclei visible) in place against the holding pipette using gentle negative pressure. Hold the micropipette loaded in place onto a micromanipulator. 7. Using the micromanipulator to guide the pipette, push the tip through the zona pellucida into the cytoplasm. 8. Using gentle positive pressure, the solution flows continuously from the pipette. The delivery into the cytoplasm is successful when the injected solution spreads to form a drop having a diameter approximately similar to the pronuclei’s one. 9. The microinjection pipette should be changed every 30–40 embryos. 10. After the injection, transfer the surviving embryos in embryo culture medium equilibrated at 37 °C, 5% CO2, and then keep them in a 37 °C humidified incubator under 5% CO2 until implantation. 3.3.3. Transfer of embryos into recipient females 1. Obtain pseudopregnant Sprague–Dawley females needed to host the microinjected embryos by mating sexually mature Sprague–Dawley females with vasectomized mature males, the night before the day of implantation. Confirm mating the
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next morning by checking for a plug. We mate 16 vasectomized males individually with females and usually obtain between 1–3 mattings. The non-mated females are used for another mating 10 days after the first attempt. 2. Transfer injected embryos into the oviduct of host females by using an embryo transfer pipette, preferably the same day as the microinjection to increase the rate of implantation. In general, no more than 24 embryos are transferred bilaterally into the oviduct. 3.4. Analysis of DNA mutations from rat tissues (Fig 3) 3.4.1. Analysis of DNA from rat tissues from 10-days-old rat 1. Harvest short tail biopsies (6 mm) 10-days-old rat. 2. Add 500 ll tissue digestion buffer to the tail biopsies in a 1.5 ml tube. 3. Incubate at 56 °C for overnight. 4. Centrifuge 18,000g at for 1 min. 5. Transfer lysate to a new microcentrifuge tube. 6. Add 500 ll isopropanol. 7. Invert tubes 20–30 times until nucleic acids precipitate. 8. Centrifuge at 18,000g for 10 min. 9. Wash nucleic acid pellet with 1 mL 70% ethanol. 10. Allow pellet to dry for 5 min. Do not dry the pellet to completion. 11. Dissolve the pellet in 100 ll TE for >15 min. 3.4.2. Analysis of gene modifications by PCR (Fig.3) 1. Design oligonucleotides that will amplify PCR products comprised between 300 and 800 bp of the region of interest from rat genome. 2. Optimize PCR conditions so that only one specific product is obtained using high-fidelity polymerase. PCR amplify the region of interest from the crude nucleic acid preparation. 3. Electrophorese the sample on a 10% polyacrylamide gel at 10– 15 V/cm or freeze at 20 °C for later analysis. 4. If the deletions are large, the visualization of PCR products on gel may allow identifying some mutated founders but all animals should be analyzed using the T7 nuclease assay and sequencing. 3.4.3. Enzymatic mismatch cleavage with T7 endonuclease I assay and sequencing 1. Perform enzymatic mismatch cleavage with T7 endonuclease I assay as described in point 3.2.
Fig. 3. T7 endonuclease I assay of rat DNA. Tails’ biopsies DNA from 4 different rats after microinjection of the same TALE nuclease were analyzed using the T7 endonuclease I assay. Rat N° 2 shows smaller bands after T7 endonuclease I treatment, indicative of a mutation.
2. The products are separated by electrophoresis to visualize evidence of T7 nuclease cleavage. The presence of mismatches is visualized by the generation of lower molecular weight bands (Fig. 3).
3.4.4. Confirmation of mutations by sequencing of PCR products 1. PCR products could be direct sequenced or cloned via TA cloning (i.e. TOPO PCR cloning system (follow the kit manufacturer’s instructions) and the presence of mutations is visualized by comparison with wild-type sequences. 2. If more than one mutation is present in the same founder animal, PCR products should be cloned in a sequencing plasmid and 10–20 bacteria colonies sequenced independently.
3.5. Results The results obtained with 4 TALE nucleases for 4 different gene targets are depicted in Table 1. We first generated rats with mutations in the IgM locus microinjecting the TALE nucleases in DNA or mRNA forms at different concentrations [22] as we had previously done for using ZFNs [19]. At the highest concentration used (10 ng/ll) we observed a toxic effect on survival of zygotes after microinjection and more obviously in birth rates and most importantly in overall mutation efficiency (mutated newborns/microinjected embryos since the number of mutated newborns was low. A key point is that the mutation rate was higher with TALE nucleases mRNA vs. DNA (4.02–10.1% vs. 0–1.81%, respectively).
Table 1 Analysis of mutations by TALE nucleases after microinjection in rat embryos. Target/construct (strain)
Route/dose (ng/ ll)
Surviving embryo rate
Birth rate⁄
Control plasmid DNA (SD) IgM/DNA (SD)
PNI/2
72.5
14.5
1.42
PNI/10 PNI/2 PNI/0.4 IC/10 IC/4 IC/0.8 IC/10 IC/2 IC/0.4 IC/5 IC/5
59 64 73 72 73 82 62.5 77 74 83 79.2
7.83 22.5 3.17 9.9 14.2 21.4 3.98 14.03 14.6 22.7 20.1
1.81 1.69 0 6.9 10.1 4.02 3.4 5.7 2.28 2.84 11.1
IgM/mRNA (SD)
X/mRNA (SD)
Y/mRNA (SD) Z/mRNA(SD)
Mutation rate⁄
% biallellic mutations
% Ins/ % Del
Size Ins/Del (%
