Animal experiments were carried out according to the guidelines of the ethics committee of Guangdong Provincial People’s Hospital. In this study, we used d/d and Pax7-CRIPSR mutant, strain tm (Pax74751V6D20/4754V6D20)LABF axolotls (Ambystoma Mexicanum) (Nowoshilow et al., 2021 (link)), aging between 2.5 and 16 months for analysis. Axolotl larvae were kept individually in plastic containers with a change of fresh tap water once a day and fed artemia daily. Axolotls were anaesthetized with 0.01% ethyl-p-aminobenzoate (Benzocaine; Sigma) solution prior to amputation, imaging, EdU injection and sample collection. Amputation was carried out on right forelimb of axolotl larvae (about 3-month-old, except for one bone regeneration experiment, 6-month-old animals were used) by cutting at the mid-upper arm followed by trimming of the bone to allow proper blastema formation. Bright-field axolotl limb images were obtained using an Olympus stereomicroscope. Samples of uninjured limbs or limb regenerates were collected at the stages indicated for analysis. If applicable, a single pulse of EdU (at a dose of 10 mg/kg body weight) was introduced into axolotls by intraperitoneal injection, and kept for 3 h prior to sample collection. All samples collected were fixed in 1 × MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4 and 3.7% formaldehyde) for further analysis, unless specified.
Ethyl p aminobenzoate benzocaine
Manufactured by Merck Group
Sourced in Germany
Ethyl p-aminobenzoate, also known as benzocaine, is a laboratory reagent used as a local anesthetic. It is a colorless crystalline solid with a characteristic odor. Benzocaine is primarily utilized as an active ingredient in topical anesthetic formulations and has various applications in the pharmaceutical and medical industries.
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Lab products found in correlation
Tricaine methanesulfonate ms 222, by Merck Group (3 mentions)
Stereomicroscope, by Olympus (1 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Dnaeasy kit, by Qiagen (1 mentions)
Topo cloning vector, by Thermo Fisher Scientific (1 mentions)
Mmessage t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions)
Ethyl 3 aminobenzoate methanesulfonate tricaine, by Merck Group (1 mentions)
10 protocols using ethyl p aminobenzoate benzocaine
1
Axolotl Forelimb Regeneration Protocol
2
Fish Welfare in Experimental Procedures
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All fish were handled according to the European Union regulations concerning the protection of experimental animals (Dir 2010/63/EU). Experimental protocols were approved by the Animal Experiments Inspectorate (AEI), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2020-15-0201-00768). Broodstock was anaesthetised individually before tagging, biopsy, and stripping of gametes, and euthanised after stripping (females) or at the end of the experiment (males) by submergence in an aqueous solution of ethyl p-aminobenzoate (benzocaine, 20 mg/L, Sigma Aldrich, Germany) [29 (link)]. Larvae were anaesthetised and euthanised using tricaine methanesulfonate (MS-222, Sigma Aldrich, Germany) at a concentration of 7.5 and 15 mg/L, respectively [29 (link)].
3
Ethical Handling of Eels and Larvae
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Eel in all stages were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 2010/63/EU). The experimental protocols were approved by the Animal Experiments Inspectorate of the Danish Ministry of Food, Agriculture and Fisheries (permit number: 2020-15-0201-00768). Eels were anesthetized in relation to tagging, biopsy, and stripping of gametes, and euthanized after stripping (females) or at the end of the experiment (males) by submergence in an aqueous solution of ethyl p-aminobenzoate (benzocaine, 20 mg/L, Sigma Aldrich, Germany). Larvae were anesthetized and euthanized using tricaine methanesulfonate (MS-222, Sigma Aldrich, Germany) at a concentration of 7.5 and 15 mg/L, respectively.
4
Animal Welfare in Fish Research
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All fish were handled according to the European Union regulations concerning the protection of experimental animals [19 ]. Experimental protocols were approved by the Animal Experiments Inspectorate (Glostrup, Denmark), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2020-15-0201-00768). Broodstock was anaesthetised individually before tagging, biopsy, and stripping of gametes, while euthanised after stripping (females) or at the end of the experiment (males) by submergence in an aqueous solution of ethyl p-aminobenzoate (benzocaine, 20 mg/L, Sigma Aldrich, Darmstadt, Germany). Larvae were anaesthetised and euthanised using tricaine methanesulfonate (MS-222, Sigma Aldrich, Darmstadt, Germany) at a concentration of 7.5 and 15 mg/L, respectively.
5
CRISPR-Cas9 Mediated Genome Editing in Newts
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Cas9 mRNA and gRNA were synthesized using mMESSAGE T7 ULTRA Transcription Kit (Invitrogen) and MEGAshortscript T7 Transcription Kit (Invitrogen), respectively. Genomic DNA was extracted with Qiagen DNAeasy kit. The PCR products were cloned into TOPO cloning vector (Invitrogen). Individual clones were sequenced with T7 primer (Supplementary Table 9 ).
Single-cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications60 (link), 61 (link). Briefly, 500 pg Cas9 RNA and 100 pg gRNA were mixed into 5 nl and injected into freshly laid single-cell-stage embryos. The animals were raised according to60 (link). The screening of F0 animals was done by both genotyping (see above) and phenotype characterization, including pigmentation analysis and immunohistochemistry of limbs/tails, according to39 (link), 40 (link). The Pax7−/− and Tyr−/− F1 animals were produced by crossing between adult F0 animals. The larvae or post-metamorphic newts were anaesthetized within 0.01% or 0.1% ethyl-p-aminobenzoate (benzocaine; Sigma) prior to imaging and tissue collection. Newts utilized for this study were processed according to Swedish Board of Agriculture animal ethical regulations (N429/12).
Single-cell fertilized eggs were obtained by either natural or induced breeding and injected according to previously published protocols with modifications60 (link), 61 (link). Briefly, 500 pg Cas9 RNA and 100 pg gRNA were mixed into 5 nl and injected into freshly laid single-cell-stage embryos. The animals were raised according to60 (link). The screening of F0 animals was done by both genotyping (see above) and phenotype characterization, including pigmentation analysis and immunohistochemistry of limbs/tails, according to39 (link), 40 (link). The Pax7−/− and Tyr−/− F1 animals were produced by crossing between adult F0 animals. The larvae or post-metamorphic newts were anaesthetized within 0.01% or 0.1% ethyl-p-aminobenzoate (benzocaine; Sigma) prior to imaging and tissue collection. Newts utilized for this study were processed according to Swedish Board of Agriculture animal ethical regulations (N429/12).
6
Eel Anaesthesia and Handling Protocol
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All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (EU Dir 2010/63) and the Animal Experiments Inspectorate (AEI), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2015-15-0201-00696). Briefly, broodstock eels were anesthetized using ethyl paminobenzoate (benzocaine, 20 mg L-1, Sigma Aldrich, Germany) before tagging and handling. Larvae were anesthetized prior to handling and euthanized prior to sampling by using tricaine methanesulfonate (MS-222, Sigma Aldrich, Germany), at a concentration of 7.5 and 15 mg L-1, respectively.
All efforts were made to minimize animal handling and stress. Permission to use 14C labelled FA was obtained by The Danish Health Board, The State Institute of Radiation Protection.
All efforts were made to minimize animal handling and stress. Permission to use 14C labelled FA was obtained by The Danish Health Board, The State Institute of Radiation Protection.
7
Anesthesia of Axolotl Specimens
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Wildtype axolotls (Ambystoma mexicanum) were obtained from the Ambystoma Genetic Stock Center (University of Kentucky, Lexington, KY) and were housed in standard conditions at 16°C in Holtfreter’s solution. Adult animals, 15–18 cm in length, were used for all experiments. Anesthesia was achieved by immersion in 0.03% ethyl-p-aminobenzoate (benzocaine; Sigma-Aldrich, St. Louis, MO) or 0.1% ethyl 3-aminobenzoate methanesulfonate (tricaine; Sigma-Aldrich). All animal experiments performed were approved by the Harvard University IACUC and were in accordance with institutional and federal guidelines.
8
Experimental Maturation of Female European Eels
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Experimental maturations were conducted on farmed female European eels, at a DTU Aqua research facility at Lyksvad Fishfarm, Vamdrup, Denmark. All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (Dir 86/609/EEC). Eel experimental protocol was approved by the Animal Experiments Inspectorate (AEI), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2010/561-1783). This study began in 2010.
Female European eels used in this study were at the pre-pubertal stage prior to experiments. As eels undergo a natural fasting period from the pre-pubertal silver stage to the end of the sexual maturation, they were not fed during treatment. All eels were anesthetized using ethyl p-aminobenzoate (benzocaine; Sigma-Aldrich, Germany), before tagging, handling and sacrifice. All efforts were made to minimize animal handling and stress.
Female European eels used in this study were at the pre-pubertal stage prior to experiments. As eels undergo a natural fasting period from the pre-pubertal silver stage to the end of the sexual maturation, they were not fed during treatment. All eels were anesthetized using ethyl p-aminobenzoate (benzocaine; Sigma-Aldrich, Germany), before tagging, handling and sacrifice. All efforts were made to minimize animal handling and stress.
9
Eel Anaesthesia and Handling Protocol
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All fish were handled in accordance with the European Union regulations concerning the protection of experimental animals (EU Dir 2010/63) and the Animal Experiments Inspectorate (AEI), Danish Ministry of Food, Agriculture and Fisheries (permit number: 2015-15-0201-00696). Briefly, broodstock eels were anesthetized using ethyl paminobenzoate (benzocaine, 20 mg L-1, Sigma Aldrich, Germany) before tagging and handling. Larvae were anesthetized prior to handling and euthanized prior to sampling by using tricaine methanesulfonate (MS-222, Sigma Aldrich, Germany), at a concentration of 7.5 and 15 mg L-1, respectively.
All efforts were made to minimize animal handling and stress. Permission to use 14C labelled FA was obtained by The Danish Health Board, The State Institute of Radiation Protection.
All efforts were made to minimize animal handling and stress. Permission to use 14C labelled FA was obtained by The Danish Health Board, The State Institute of Radiation Protection.
10
Tissue-specific immune gene expression in European eel
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The tissue specific expression of the studied immune genes was investigated using three immature female European eels at the yellow eel stage raised from the glass eel stage to a size of 58 ± 1.6 cm and weight of 470 ± 39.7 g at a Danish commercial fish farm (Stensgård Eel Farm A/S).
The eels were euthanized by submersion in an aqueous solution of ethyl p-aminobenzoate (benzocaine) at 20 mg L -1 (Sigma-Aldrich, Missouri, USA) and organ tissue samples dissected from hind-gut, gills, head kidney, kidney, liver, skin, spleen, whole brain, heart, and muscle. Samples were stored in RNA-later at -80°C until further use. For further processing see 2.4.
The eels were euthanized by submersion in an aqueous solution of ethyl p-aminobenzoate (benzocaine) at 20 mg L -1 (Sigma-Aldrich, Missouri, USA) and organ tissue samples dissected from hind-gut, gills, head kidney, kidney, liver, skin, spleen, whole brain, heart, and muscle. Samples were stored in RNA-later at -80°C until further use. For further processing see 2.4.
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