4.2. Animals and Animal Procedures

All animal procedures that were used in this study were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center (UTSW) and were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Soat1^+/− mice on a C57BL/6J background were purchased from the Jackson Laboratory (B6.129S4-Soat1^tm1Far/Pgn; stock #007147; Bar Harbor, ME, USA). The targeted mutation resulted in Soat1-null mice which have no SOAT1 protein detected by immunoblotting samples from preputial gland, ovaries, and adrenals [32].

The animal colonies were maintained and bred in the Animal Research Center (ARC) of UTSW under constant supervision of ARC staff members (technicians and veterinarians) on a 12-h light/dark cycle with ad libitum access to food and water on the 2016 Teklad global 16% protein rodent diet (from Envigo, Indianapolis, IN, USA).

Mice were genotyped using three primers described in the Jackson Laboratory protocol #24675 (all sequences 5′→3′): Primer 18,958 (TGC TGA CGT CTT CCT GTG TC, common), primer 18,959 (GAG CTG TTG GG AGT AGG TG, wild-type reverse), and primer oIMR6218 (CCT TCT ATC GCC TTC TTG ACG, mutant reverse), which all provided the expected bands 400 bp (for mutant) and 240 bp (for wild type).

Evaluation of mouse ocular features was conducted using a slit lamp model BQ 900 (from Haag–Streit USA, Inc., Mason, OH, USA) as described recently [14]. TP samples were collected for analyses using a Zeiss Stemi 508 Stereo microscope (Carl Zeiss, Oberkochen, Germany) as described earlier [14]. Treated mice were euthanized using inhalant isoflurane followed by cervical dislocation. Briefly, four TP from each mouse were excised from the eyelids, dissected free from epidermis, and placed in HPLC-style glass sample vials filled with ~0.5 mL of chloroform:methanol = 2:1 solvent mixture (v/v; CM21) for overnight extraction in a fridge. Then, the lipids were repetitively extracted with 3 × 1 mL of the same solvent mixture, the extracts were combined, the solvent was evaporated at 37 °C under a gentle stream of nitrogen, and the remaining lipid material was re-dissolved in iso-propanol (between 0.2 and 0.5 mL per 4 TP). The samples were sealed and stored at −20 °C until analyses. Samples of meibum were obtained by expressing the secretion from the orifices, collecting the samples with a microspatula, and dissolving the specimens in the CM21 solvent mixture. For RP UPLC/APCI MS analyses, the CM21 mixture was evaporated and the samples were re-dissolved in iso-propanol as described above. Five Soat1^−/−, seven Soat1^+/−, and seven WT mice were tested. Statistical aspects of the analyses (necessary numbers of biological samples and technical replicas, reproducibility of the analyses, methods of sample quantitation, etc.) are described in detail in our recent publications [7,12,14,21] and are not repeated here to avoid duplication in reporting.