Hydrodynamic Tail Vein Injection for Liver Tumor Model

For HDTVi, vectors for HDTVi were prepared using the EndoFree-Maxi Kit (Qiagen) and resuspended in a sterile 0.9% NaCl solution/plasmid mix containing 10 μg of pX330-p53 (Addgene 59910) or pT3-N90-beta-catenin (Addgene 31785), and 10 μg of CMV-SB13 Transposase. CRISPR-Cas9 vector system carrying sgRNAs targeting Trp53 together with the Sleeping Beauty Transposon system overexpressing CTNNB1-N90 vector in sterile saline constituted a total volume of 10% of the mouse body weight were injected into the lateral tail vein of 6-week-old C57BL/6 J mice in 6–8 s^{15 (link),16 (link)}. HDTVi-induced tumors were harvested 3 weeks after HDTVi. For staining and lineage tracing assays, 3–6 mice (male and female) were used per group. 6-week-old mice male and female mice were purchased from The Jackson Laboratory, including C57BL/6J mice (JAX stock, #000664), Acta2-iCre-PolyA (Gem Pharmatech, #T036743), Cd36em1(flox) (Shanghai Model Organisms Center, #NM-CKO-200086), Mifem1(flox) (Shanghai Model Organisms Center, #NM-CKO-2110274), Lratem1(2A-Cre) (Shanghai Model Organisms Center, #NM-KI-190097), R26-CAG-LSL-tdTomato (Shanghai Model Organisms Center, # NM-KI-225042). All animals were housed in a pathogen-free facility with 24-h access to food and water. Animal experiments in this study were approved by and performed in accordance with the institutional animal care and use committee at the Zhongshan hospital, Fudan University. Mice were euthanized by cervical dislocation under anesthesia.