Mouse research protocols and transgenic lines reveal that venousization enables angiogenesis in the absence of vascular endothelial growth factor receptor signaling.
Husbandry and experimental procedures followed protocols approved by local animal ethics committees and authorities, specifically the Comunidad Autónoma de Madrid and Universidad Autónoma de Madrid under permits CAM-PROEX 164.8/20, PROEX 293.1/22, and PROEX-288.7/24, Nature reported. Colonies were maintained in racked individual ventilation cages with dust- and pathogen-free bedding, alongside nesting and environmental enrichment materials designed to support species-specific behaviour. Housing conditions maintained ad libitum access to food and water at temperatures between 21 °C and 24 °C, relative humidity levels of 45–65%, and a 12-hour light–dark cycle.
Researchers Intercrossed Multiple Mouse Lines and Injected Cas9 into Eggs
Investigators intercrossed multiple mouse lines, including Tg(Cdh5–CreERT2)44, Tg(iSuRe–HadCre)14, Vegfr1flox, Vegfr2flox, and Vegfr3flox alleles, alongside Nr2f2flox, iMb2–Mosaic16, Apln–FlpO15, and the iFlpMosaic lines consisting of R26-iFlpMTomato-2A-Cre/MYFP and Tg-iFlpChromatin–Mosaic, according to Nature. To build a new Vegfr3flox allele combined with Vegfr1/2flox, researchers injected Cas9, donor DNAs carrying LoxP sites, and guide RNAs directly into mouse eggs harvested from the Vegfr1/2flox iSuRe–HadCre Cdh5–CreERT2 line. Founders bearing LoxP sites flanking exon 1 on the same chromosome were screened via PCR, while inducible deletion of Vegfr3 was verified through PCR and immunostaining directed at the VEGFR3 protein.
Adult Mice Received Intraperitoneal Injections of Tamoxifen Dissolved in Corn Oil
To induce CreERT2 activity in adult mice, 1 gram of tamoxifen from Sigma-Aldrich was dissolved in 50 millilitres of corn oil to establish a stock concentration of 20 milligrams per millilitre, with aliquots stored at −20 °C, as detailed by Nature. Animals received intraperitoneal injections of 100 microlitres from this stock, delivering a total dose of 2 milligrams per animal at 80 milligrams per kilogram. Pups requiring recombination activation received 4-OHT injections at the indicated stages at a dose of 40 mg kg−1, whereas double mutants involving VEGFR1/2 or VEGFR2/3, as well as triple mutants involving VEGFR1/2/3, received 4-OHT administered to pups at 15 mg kg−1. During embryogenesis, pregnant dams were induced at embryonic stages E10.5 and E12.5 using 0.25 milligrams of 4-OHT supplemented with 0.125 milligrams of progesterone.
Lewis Carcinoma Cells Were Cultured and Processed for Single Cell Sequencing
Lewis carcinoma cells obtained from ATCC were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 2 millimolar glutamine under 37 °C and 5% CO2 conditions, according to Nature. Single-cell suspensions were evaluated for viability and concentration using a Countess 3 Automated Cell Counter prior to encapsulation into emulsion droplets via the 10x Genomics Chromium Controller system. Library preparation and sequencing took place at the CNIC Genomics Unit using 20,000 to 50,000 cells per port on an Illumina NextSeq 2000 platform running software version 1.7.2. The resulting single-cell RNA sequencing data were aligned and quality-controlled by the CNIC Bioinformatics Unit using Cell Ranger versions 8 and 9.
