Stock No: 006775
Protocol 13662: Pyrosequencing Assay - Foxp3<sf>
Version 6.0

Notes

The Foxp3 gene is X-LINKED. Males with the mutant allele are hemizygous mutants.

Hemizygous mutant males die 16-25 days after birth.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = AA insertion
Wild type = NO AA insertion

Sequence

Insertion of two adenosine residues into exon 8, resulting in a 2 bp shift in the reading frame. This allele is predicted to produce a truncated protein lacking the carboxy-terminal forkhead domain. (J:66695)

GAAAGGAATGAGAAGAAGGAAGATAAGCGATGAGGGAGGAGATAGGGTGAAA
GTGGTCTGTATGTATTACATACATGTACAAAATTGTCTAAAAACAAGTTTAACTAAT
AAGAAAATACAAACTAATGTTTGAAAGGCTACAATGAAATGACAAGCTTAAGTGT
CTCGATTACCACACCCCTCCCAACCCCTCAGGCCTCAATGGACAA(aa)GAGCT
CTTGCTGCATCGTAGCCACCAGTACTCAGGGCAGTGTGCTCCCGGCCTGGTC
TGCTCCTCGGGAGGCTCCAGACGGCGGCCTGTTTGCAGTGCGGAGGCACCT
CTGGGGAAGCCATGGCAATAGTTCCTTCCCAGGTCAGTGGAGTCCACACCCC
AGTGCCAGGGGGTACAAAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9182 GGC CTC AAT GGA CAA
poIMR0139 AAC CCC TCA GGC CTC AAT
poIMR0140 Fluorophore GGA GCA CAC TGC CCT GAG TA

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
poIMR0139 0.50
poIMR0140 0.50
Glycerol 5.00
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.