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 and purple dye.

• 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.

Measure DNA quantity/quality with Nanodrop.

• yield was 60 - 1100 ng/ul genomic DNA.

PCR

PCR master-mix: Promega Gotaq colorless master mix.

For 50 λ reaction:

• 25 λ Gotaq
• 10 λ mixture of 5 uM forward/reverse Tyro3 primers
• 14 λ dH₂O
• 1 λ of genomic DNA

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).

Gel

2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 λ of SYBR safe 10000x while cooling). Make mini-gel with 12-tooth comb

load 10 λ of 50bp ladder; 10 λ of PCR product + 2 ul purple loading dye

run at 90V for 30 min (or 65V for 60 min for sharper bands). Red dye front migrates at about 370bp, similar to bromophenol blue.

PCR clean-up

E.Z.N.A. Cycle Pure Kit (V-spin) yields 30 λ in dH₂O [ because worried about pH of dH₂O, now trying kit elution buffer which I think is Tris-Cl pH 8.5]

Measure DNA quantity/quality with Nanodrop.

• yield was 20 - 65 ng/λ clean PCR product

Re-Amplification

To get higher concentration of DNA for digest, repeat PCR using 1 λ of clean PCR product and clean up.

• yield was xxx - xxx ng/λ clean re-amplified PCR product

(could also pool repeated original PCR Rxns from genomic DNA, but that seems inefficient. Re-amplifying risks PCR artifacts/mutations, though ...)

Digestion

• 1 λ NlaIV (2 units)
• 5 λ rCutSMart Buffer 10x
• 1 ug clean Tyro 3 PCR product
• H₂O to 50 λ

Incubate at 37 C for 1 hour, heat inactivate 65 C for 20 min.

NEBiolabs says to mix digest with purple dye with 0.5% SDS to separate protein from DNA. SDS may interfere with SYBR Safe if stain mixed in the gel, so may need to post-stain the gel.