Sample 4
Protocols: VaviCre (B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J, Jackson laboratory) heterozygous male mice were bred with female Kdm6afl/fl (B6;129S-Kdm6atm1.1Kaig/J, Jackson laboratory) homozygous mice to establish VaviCreKdm6afl/f offspring with a haematopoietic specific KDM6A knockout (referred to in this manuscript as Kdm6aΔ-Haem mice). Control mice (referred to in this manuscript as Kdm6aWT mice) were littermates lacking the VaviCre transgene. Phenotyping experiments included both male and female mice. The animals were sedated using isoflurane, and pain relief was provided through an intraperitoneal injection of Buprenorphine (0.1 mg/kg body weight) along with an intercostal nerve block using Bupivacaine (1 mg/kg body weight, 0.25% concentration). For postoperative pain management, Buprenorphine and Carprofen (5 mg/kg body weight) were administered every 12 or 24 hours for three days. To prevent infection after surgery, Ampicillin (100 mg/kg body weight) was added to the drinking water. The myocardial infarction (MI) was induced by permanently ligating the left anterior descending coronary artery while the animals were under mechanical ventilation. urine IL-1β was inhibited post-MI via intraperitoneal injection of InVivoMAb anti-mouse/rat IL-1β antibody (Armenian hamster IgG isotype, clone B122, BE0246, BioXcell) at a dosage of 10mg/kg. Control animals were given InVivoMAb polyclonal Armenian hamster IgG (BE0091, BioXcell) at the same dosage. Injection volume was 100μl and the dilutant was InVivoPure pH 7.0 Dilution Buffer (BioXcell). Mice were given a total of 4 injections post-MI; the first one 2 hours post-MI and then three more at a weekly basis before termination of the experiment at 4 weeks post-MI. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's guidelines. RNA content and purity were assessed by spectrophotometry (NanoDrop Technologies). mRNA expression was quantified by RT-qPCR using total RNA, which was reversed transcribed by MuLV reverse transcriptase (Thermo Fisher Scientific) and random hexamer primers (Thermo Fisher Scientific) cDNA synthesis was performed according to the protocol: (Primer annealing) 65°C x 5 min, (Primer elongation) 25°C x 10 min, (Reverse transcription) 37°C x 50 min, (Enzyme denaturation) 70°C x 15 min. Expression levels of mRNA were detected by using Fast SYBR Green Applied Biosystems and an Applied Biosystems Viia7 machine. Cycling conditions on the machine were as follows: (Hold) 95°C x 30 sec, (PCR) 95°C x 1 sec, 60°C x 20 sec repeated for 40 cycles, (Melt curve) 95°C x 15 sec, 60°C x 60 sec, 95°C x 15 sec.  Expression levels were additionally detected using the Taqman based system using the TaqMan™ Fast Advanced Master Mix. Cycling conditions on the machine were as follows: (Hold) 95°C x 20 sec, (PCR) 95°C x 1 sec, 60°C x 20 sec repeated for 40 cycles.  Relative gene expression was calculated with the QuantStudio Real-Time PCR software (version 1.3) using 2-ΔCt or 2-ΔΔCt. Rplp0 was used as housekeeping gene. SYBR green sequences and Taqman probe IDs can be found in the Supplementary Table.
strain
VaviCre +/- Kdm6afl/f
individual
Female_IgG_13_S13
organism
Mus musculus
organism
Mus musculus
collection date
not collected
scientific_name
Mus musculus
common name
house mouse
organism part
heart
developmental stage
adult
genotype
VaviCre +/- Kdm6afl/f (KDM6AΔ-Haem)
isolate
not applicable
biological sex
female
genetic modification
IgG
age
16
geographic location (country and/or sea)
not collected