title: REPS3
organism: Sus scrofa
description: Protocols: Prior to euthanasia, anesthesia was induced using Telazol (4 mg/Kg) and Xylazine (2 mg/kg) followed by Propofol (3-7 mg/kg) and then maintained with Isoflurane (1-3 MAC) following intubation. Eyes were protected using an eye lubricant. Both limbs were shaved and scrubbed with Chlorhexidine and 70% alcohol until visibly clean, followed by a ten minute evaporation period. Hoofs were covered with unsterile gloves. Animals were then transferred into the adjacent operating room, placed supine on a heating mat, and secured on the operation table. Animal health and anesthesia depth were maintained by monitoring respiratory rate, oxygen saturation, electrocardiogram, blood pressure, and body temperature. The surgical limb and ipsilateral lower body were then scrubbed three times using Betadine. Hoofs were covered with a sterile glove and secured using a sterile elastic wrap. One layer of sterile towels was placed around the surgical area, followed by a layer of sterile drapes, leaving only the surgical limb exposed during the procedure. Animals were euthanized during deep anesthesia using an intravenous injection of a solution containing pentobarbital sodium and phenytoin sodium (Beuthanasia-D, 0.1ml/kg). Death was confirmed by the absence of blood pressure and heart sounds prior to obtaining the tissue samples. The medial meniscus of the surgical leg along with the attached synovium and capsule were excised. This tissue sample was divided according to the frontal plane through the center of the pars intermedia. The synovium posterior of this plane was harvested for immediate homogenization. Synovium samples were immediately placed in 2 ml Lysing matrix S tubes (MP Biomedical) and homogenized in 1 ml TRIzol (Life Technologies) using a FastPrep-24 Instrument (MP Biomedical). Samples were then frozen in liquid nitrogen, transferred on dry ice and stored at -80°C until RNA isolation. Forty-two adolescent Yucatan minipigs (Sinclair BioResources, Columbia MO) were allocated to unilateral ACL transection surgery (n=36) or no surgery (INTACT, n=6). Animals receiving ACL transection surgery were allocated to euthanasia and outcome assessment at 1 week (1W, n=18) or 4 weeks (4W, n=18) after surgery. Within each time-point 6 of 18 animals were allocated to no treatment following transection, 6 of 18 to immediate ligament reconstruction surgery, and 6 of 18 to immediate ligament repair surgery. A computer based random permutation stratified for sex determined each animal's group allocation and side of unilateral surgery with an equal number of males and females in each group. While the animals were under anesthesia, a medial arthrotomy was performed and the fat pad partially resected to expose the ACL. The ACL was transected between the proximal and middle thirds of the ligament. A clinical exam was performed to verify complete ACL transection. In the animals assigned to receive ACL reconstruction surgery, a fresh-frozen bone-patellar-tendon-bone allograft, which was harvested from an age-, weight-, and sex-matched donor, was implanted as previously described (Murray and Fleming. AJSM 2013). In the animals assigned to ACL repair surgery, an extracellular matrix scaffold in combination with autologous blood was implanted as previously described (Murray and Fleming. AJSM 2013). Knees were thoroughly irrigated with 500 mL of normal saline following ACL transection in the group that did not receive further surgical treatment or following bone tunnel placement in those that received ACL reconstruction or repair. All incisions were closed in layers. No postoperative immobilization was used. Total RNA was extracted from the synovial membranes through homogenization, phenol-chloroform separation and on-column purification using the PureLink™ RNA Mini Kit (Life technologies, Carlsbad, CA). RNA samples were treated with DNAse I (PureLink™ DNase Set, Life technologies, Carlsbad, CA) and assessed with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The samples were then enriched for polyA+ messenger RNA, reverse transcribed with random hexamers, ligated with indexed adapters and amplified with 17 cycles of PCR using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-04-21T17:01:57Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-04-11T05:20:54Z
attributes.tag: External Id
attributes.value: SAMEA1100181
attributes.tag: INSDC center name
attributes.value: Boston Children's Hospital, Harvard Medical School
attributes.tag: INSDC first public
attributes.value: 2018-04-21T17:01:57Z
attributes.tag: INSDC last update
attributes.value: 2018-04-11T05:20:54Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6664:REPS3
attributes.tag: age
attributes.value: 17.7
attributes.tag: breed
attributes.value: Yucatan
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: pig
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: injury
attributes.value: anterior cruciate ligament transection surgery
attributes.tag: organism part
attributes.value: synovial membrane
attributes.tag: sample name
attributes.value: E-MTAB-6664:REPS3
attributes.tag: sampling site
attributes.value: perimeniscal medial femorotibial joint
attributes.tag: scientific_name
attributes.value: Sus scrofa
attributes.tag: sex
attributes.value: female
attributes.tag: treatment
attributes.value: ligament repair surgery (BEAR)