Difference between revisions of "Anorexia anx/anx mutant mice"

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Anorexic anx/anx mice [https://www.jax.org/strain/000624 Jackson Lab strain 000624].
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''Anorexia'' anx/anx mice [https://www.jax.org/strain/000624 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).
 
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).
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== Possible genes affected ==
 
== Possible genes affected ==
  
Lindsfor 2011: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1)  
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Lindfors 2011 PMID 22025706: NADH dehydrogenase (ubiquinone) 1a-subcomplex (Ndufaf1)  
  
Kim 2017: tyrosine kinase receptor Tyro3  which they conclude is not the anx-mutation but a strain specific modifier of anx-phenotypes  
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Kim 2017 PMID 28093506: tyrosine kinase receptor Tyro3  which they conclude is not the anx-mutation but a strain specific modifier of anx-phenotypes  
  
 
== Abnormalities Table ==
 
== Abnormalities Table ==
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== Tyro3 Genotyping ==
 
== Tyro3 Genotyping ==
  
TYRO3 protein tyrosine kinase 3 (Tyro3) mutation in anx/anx eliminatees a NlaIV restriction site.
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TYRO3 protein tyrosine kinase 3 (Tyro3) mutation (C19T-Tyro3 mutation) in anx/anx eliminates a NlaIV restriction site.
  
* primers 5′-GATGGCGCTGAGGCGGAGCATG and 5′-CGCGGC- CGGAGGTCTGGCAG ; annealing temperature 72C
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* primers forward 5′-GATGGCGCTGAGGCGGAGCATG and reverse 5′-CGCGGCCGGAGGTCTGGCAG ; annealing temperature 72C
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* predicted PCR product size: 280bp equal to [https://www.ncbi.nlm.nih.gov/nucleotide/1877089967?from=119630270&to=119630549&report=gbwithparts Mus musculus strain C57BL/6J chromosome 2, GRCm39], where forward primer is 119630270-119630291 and reverse primer is 119630549-119630530
  
 
* subsequent digestion with NlaIV  
 
* subsequent digestion with NlaIV  
  
* wildtype -> 175 bp fragment, anx/anx -> 250 bp product
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* wildtype -> 175 bp fragment, anx/anx -> 250 bp product (eyeballing from Kim 2017)
  
[[File:anx_pcr_kim_2017.png|800px|thumb]]
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[[File:anx_pcr_kim_2017.png|800px|border]]
  
  
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* 2024-1-17 Tyro3 primers ordered from FSU BIO Core  
 
* 2024-1-17 Tyro3 primers ordered from FSU BIO Core  
  
* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product%20Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product%20Information 50bp ladder]
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* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product-Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product-Information 50bp ladder] and purple dye.
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* 2024-6-6 [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega GoTaq colorless master mix] ordered from Fisher (suspect green master mix interferes with digest)
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===DNA extraction from tails/spleens===
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[https://omegabiotek.com/product-category/genomic-dna/cultured-cells-and-tissue/ E.Z.N.A. Tissue DNA Kit] yields 200 ul in elution buffer.
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Measure DNA quantity/quality with Nanodrop.
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===PCR===
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PCR master-mix: [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega Gotaq colorless master mix].
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For 50 ul reaction:
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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).
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===Gel===
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2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 ul of SYBR safe 10000x)
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load 10 ul of 50bp ladder; 10 ul of PCR product + 2 ul purple loading dye
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run at 90V for 30 min
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===PCR clean-up===
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[https://omegabiotek.com/product/pcr-clean-up-kit-e-z-n-a-cycle-pure/ E.Z.N.A. Cycle Pure Kit (V-spin)] yields 30 ul in dH<sub>2</sub>0
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===Digestion===

Latest revision as of 09:58, 24 October 2024

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

Abnormalities Table

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

Anx pcr kim 2017.png


Genotyping 1998 mice

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

DNA extraction from tails/spleens

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

Measure DNA quantity/quality with Nanodrop.

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).


Gel

2% agarose in TAE (2 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 ul of PCR product + 2 ul purple loading dye

run at 90V for 30 min

PCR clean-up

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

Digestion