Crispr cas9 technique

C57BL/6N wild-type male mice were obtained from the OrientBio and Trim40^-/- male mice were generated on C57BL/6N background using CRISPR/Cas9 technique from Cyagen Biosciences. CRISPR guide RNA (gRNA) was used to target exon 1 region in the Trim40. Genotypes of mice were investigated using genomic DNA PCR. All mice were maintained in the specific pathogen-free facility of the Yonsei Laboratory Animal Research Center at Yonsei University according to Korean Food and Drug Administration. All mice were housed on a 14-hour light and 10-hour dark cycle with 18–23 °C and 40–60% humidity and were maintained in ventilated cages under standard housing conditions with access to food and water ad libitum. No more than 5 mice were housed in cages and all mice used in this study were appropriately monitored daily changes in body weight, food or water intake, and physical appearance. At experimental endpoints, mice were euthanized with CO₂ inhalation for at least 5 min until breathing and heartbeat stopped. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei University (YLARC, No. IACUC-202011-1177-01, IACUC-202107-1302-02, IACUC-202107-1302-03, IACUC-202204-1447-01, IACUC-202204-1448-01, IACUC-202209-1541-01 and IACUC-A-202209-1542-01).

Six to eight weeks old female BALB/c mice (Guangdong Medical Laboratory Animal Center) and TLR4 knockout C57BL/6 mice generated using CRISPR/Cas9 technique (Cyagen Biosciences) were bred under standard pathogen-free conditions. For DNA vaccination, each mouse was immunized with 20 μg of endotoxin-free DNA vaccines or pVAX1 vector twice at four-week intervals intramuscularly plus electroporation as we described previously [13 (link)]. Two weeks after the last immunization, mice were euthanized for immunogenicity analysis. For tumor challenge, two weeks after the 2nd immunization, 5 × 10⁵ AB1-Gag cells were inoculated subcutaneously in the right flank of mice. The tumor size was measured each two-four days. Tumor size based on caliper measurement was calculated by the modified ellipsoidal formula, tumor size = (length × width²)/2. Mice were sacrificed at the endpoint (21 days post-challenge). The mice were raised under specific pathogen-free (SPF) conditions and were fed a normal diet.

All animal procedures were designed and performed according to the regulations of the Institutional Animal Care and Use Ethics Committee of Zhujiang Hospital. The establishment of conventional LBH- KO C57BL/6 mice was performed by Cyagen, Inc., via the CRISPR/Cas9 technique (contract ID: KOAI200813MG1) to generate F₀ founder animals; then, the wild-type (WT)/KO offspring were bred, identified, and maintained at the Animal Experiment Center of Zhujiang Hospital (Figure S1). The MI model was constructed according to our previous research [13 (link)]. All mice were sacrificed to obtain the hearts on days 2 and 3 after surgery. Each experimental group had at least four surviving mice for sampling at every time point. Paraffin-embedded tissue sections of the left ventricles were used for hematoxylin and eosin (H&E), Masson, and immunofluorescence staining (Figure S2), and the remaining peri-infarct tissues were used for western blot (WB) assays.