Generation of Endothelial Cell –Targeted Gch1 Knockout Mice
Endothelial cell–specific BH4 deficient mice and their littermates controls were generated by crossing Gch1fl/fl females with Gch1fl/fl Tie2cre male mice as described previously (Chuaiphichai et al., 2014). Mice were housed in ventilated cages with a 12-hour light/dark cycle and controlled temperature (20–22°C), and fed normal chow and water ad libitum. Female Gch1fl/fl Tie2cre mice and their Gch1fl/fl littermates (thereafter referred to as wild-type) were used for all experiments at 10 to 16 weeks. The generation and phenotyping of the knock-out model were carried out in accordance with the Animal (Scientific Procedures) Act 1986, with procedures reviewed by the clinical medicine animal care and ethical review body, and conducted under project licenses PPL 30/3080 and P0C27F69A. Mice were genotyped by polymerase chain reactions using DNA prepared from ear biopsies. For Gch1fl/fl genotyping, PCR was performed using the following primers:Gch1fl/fl -Fw 5’-GTC CTT GGT CTC AGT AAA CTT GCC AGG-3’, Gch1fl/fl -Rv 5’-GCC CAG CCA AGG ATA GAT GCA G-3’. The Gch1 floxed allele showed a 1030 bp. For Tie2cre genotyping, PCR was performed using the following primers: Tie2cre Fw 5’-GCA TAA CCA GTG AAA CAG CAT TGC TG-3’. Tie2cre Rv 5’-GGA CAT GTT CAG GGA TCG CCA GGC G-3’. The Tie2cre allele amplified as 280 bp fragment.

Timed mating

Pregnancy was achieved by mating either virgin femaleGch1fl/fl Tie2cre orGch1fl/fl (wild-type) females (aged between 10 to 16 weeks old) with a Gch1fl/fl male. Detection of a vaginal plugs indicated successful conception and was taken as 0.5 day of gestation (E0.5).