Anorexia anx/anx mutant mice

Anorexia anx/anx mice Jackson Lab strain 000624.

Mutation mapped to a 0.2 cM interval residing between the markers D2Mit133 and Jojo5 chromosome 2 (Chr 2: bp 118, 889, 896–120, 175, 1081) (Lindfors et al., 2011).

Possible genes affected

Lindfors 2011 PMID 22025706: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1)

Kim 2017 PMID 28093506: tyrosine kinase receptor Tyro3 which they conclude is not the anx-mutation but a strain specific modifier of anx-phenotypes

Tyro3 Genotyping

TYRO3 protein tyrosine kinase 3 (Tyro3) mutation (C19T-Tyro3 mutation) in anx/anx eliminates a NlaIV restriction site.

• primers forward 5′-GATGGCGCTGAGGCGGAGCATG and reverse 5′-CGCGGCCGGAGGTCTGGCAG ; annealing temperature 72C

• predicted PCR product size: 280bp equal to Mus musculus strain C57BL/6J chromosome 2, GRCm39, where forward primer is 119630270-119630291 and reverse primer is 119630549-119630530

• subsequent digestion with NlaIV

• wildtype -> 175 bp fragment, anx/anx -> 250 bp product (eyeballing from Kim 2017)

Genotyping 1998 mice

• 2024-1-17 Tyro3 primers ordered from FSU BIO Core

• 2024-1-18 NlaIV ordered from NEBiolabs, along with 50bp ladder

• 2024-6-6 Promega GoTaq colorless master mix ordered from Fisher (suspect green master mix interferes with digest)

DNA extraction from tails/spleens

E.Z.N.A. Tissue DNA Kit yields 200 ul in elution buffer.

PCR

PCR master-mix: Promega Gotaq colorless master mix.

For 50 ul reaction:

PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).

PCR clean-up

E.Z.N.A. Cycle Pure Kit (V-spin) yields 30 ul in dH₂0

Gel

1% agarose in TAE (1 g agarose in 100 ml TAE, microwave 2 min at 50 power; add 10 ul of SYBR safe 10000x)

load 10 ul of 50bp ladder, 10-20 ul of pre-digest and digest