1. Supplementary Figure S1 Generation of Nphp3G2A knock-in mice
Forward primer
Reverse primer
exon 1 2 3 4 25 26 27
1 kb
XbaⅠ
XbaⅠ
WT Nphp3
allele
ATG
probe
5´ arm
loxP
loxP
3´ arm
Targeting construct *
neor
GGCGCC
Gly Ala
XbaⅠ
Nphp3G2A
allele (flox) *
neor
Nphp3G2A
allele (after Neo deletion) * b c d
Nphp3 genotype
WT WT/G2A G2A/G2A
5 kb
A T G G G C
Met Gly
A T G G C C
Met Ala
WT WT/G2A G2A/G2A
0.5 kb
Nphp3G2A/G2A
WT allele WT
Nphp3G2A allele
Supplementary Figure S1 Generation of Nphp3G2A knock-in mice. (a) Targeting strategy to replace the second glycine of Nphp3 to alanine. Alleles produced after homologous recombination and Cre recombination are shown. Neo=neomycin resistance cassette. (b) Verification of correct gene targeting (Southern blot). (c) Sanger sequencing confirmed the homozygous c.5G>C mutation in Nphp3G2A/G2A mice but not in wild-type (WT) mice (arrows). (d) PCR-mediated genotyping of WT, Nphp3+/G2A and Nphp3G2A/G2 mice. Product lengths are 375 bp for WT and 502 bp for knock-in alleles.