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).