Clorketam 1000

In both mice and dogs, lentivector injections were performed in the muscular fascia in the left posterior paw, after a slight skin incision. For each animal, a control injection was performed in the right posterior paw (Supplementary Table 1). All mice were injected with 20 µl of vector (LentiFlash^® Cre particles or E7/HPV16‐ZsGreen ILV) under intraperitoneal anesthesia with a mix of 60–70 mg kg⁻¹ ketamine (Clorketam^® 1000, Vetoquinol) and 0.5 mg kg⁻¹ medetomidine (Medetor^®, Virbac) previously diluted in physiological serum. All dogs were injected with 50 µl lentivector (E7/HPV16‐ZsGreen ILV) under subcutaneous tranquilization with 0.01 mg kg⁻¹ acepromazine (Calmivet^®, Vetoquinol) diluted in physiological serum beforehand and 0.3 mg kg⁻¹ butorphanol (Dolorex^®, Merck). In addition, dogs received 6 mg of lidocaine (Lurocaine^® Vetoquinol), locally around the injected area, a few minutes before the lentivector administration.
The conditions of administration are described in Additional file 4: Table S1.

At the end of week 7, the animals were euthanized, after 12 h fasting, by intraperitoneal administration of ketamine (75 mg/kg, Rompun^® 2%, Bayer Healthcare SA, Kiel, Germany) and xylazine (10 mg/kg, Clorketam 1000, Vetoquinol, Barcarena, Oeiras, Portugal), followed by exsanguination by cardiac puncture. Blood samples were collected directly from the heart and centrifuged at 3000 g/10 min for serum separation. Glucose, albumin, triglycerides and cholesterol levels were determined in an autoanalyzer (Prestige 24i, Cornay PZ, Lomianki, Poland). A complete necropsy was performed in all animals, and the internal organs were macroscopically observed, collected, weighed, and preserved in 10% buffered formalin for 24 h for histopathological analysis. A veterinary pathologist blindly examined 4-µm sections of paraffin-embedded kidneys, stained with hematoxylin and eosin (H&E) under a light microscope.

Rats were anesthetized by a mixture of ketamine (Clorketam1000; Vetoquinol, France) and xylazine (Sigma Aldrich; St. Quentin Fallavier, France) at 150 mg/kg and 6 mg/kg, respectively.

Three-month-old Wt and Tg-RGN Sprague Dawley rats (Rattus norvegicus) (Charles River, Barcelona, Spain and Japan SLC, Hamamatsu, Japan, respectively) were maintained with food and water ad libitum in a constant room temperature (20 ± 2 °C) on a 12-hour cycle of artificial lighting. All experiments complied with the US National Institutes of Health guidelines⁷⁷ and the European Union rules for the care and handling of laboratory animals (Directive 2010/63/EU). The protocol for animal tissue collection was approved by the local “FCS-UBI Animal Facilities Committee”. All rats (n = 6 in each group) were euthanized under anesthesia (Clorketam 1000, Vetoquinol, Lure, France) and the testes removed. One testis from each animal was used for fluid collection, and the contralateral testis for SCs isolation and SeT culture.