Sep 19, 2024

Public workspaceNf1 loxP site verification

DOI
https://dx.doi.org/10.17504/protocols.io.261ge3ryyl47/v1
  • Greig Couasnay1,
  • Florent Elefteriou1
  • 1Baylor College of Medicine
Florent Elefteriou
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge3ryyl47/v1
External link: https://www.jax.org/strain/017639
Protocol Citation: Greig Couasnay, Florent Elefteriou 2024. Nf1 loxP site verification. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3ryyl47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 20, 2022
Last Modified: September 20, 2024
Protocol Integer ID: 74278
Keywords: Neurofibromatosis type 1, NF1, Genotying, PCR, loxP, conservation of the loxp site, flox mouse colony, loxp site verification, nf1 flox, loxp1, loss of dna recombination, identified skeletal phenotype, loxp2 site, detecting potential mutation, loxp site, potential mutation, recombinase activity, skeletal phenotype, other nf1, dna recombination, gene, nf1, phenotype, mice, dna
Funders Acknowledgements:
NIH
Grant ID: R01AR077949
Disclaimer
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The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
We have found that our Nf1 flox mouse colonies contained mice that had lost one of the two loxP sites inserted into the Nf1 gene (Parada laboratory, PMID:11297510, JAX# 017639), leading to loss of DNA recombination upon Cre-recombinase activity and loss of previously identified skeletal phenotypes. This is likely to occur in other Nf1 flox mouse colonies.
We describe here a strategy to verify conservation of the loxP sites within the Nf1 allele and to sequence the loxP1 and loxP2 sites for detecting potential mutations or deletions. This may be done if the expression of Nf1 (qPCR) is not reduced in CreTg/+; Nf1 flox/flox tissues of interest.
Attachments
Icon for Exon 31_32 and loxP verif primers.pdf
Exon 31_32 and loxP ...
1.3MB
Materials
Heater (DNA denaturation/Lysis)
PCR machine
Agarose gel electrophoresis system (DNA separation)
Gel documentation system (DNA amplicon visualization and picture)

Protocol materials
ReagentSpecific Primers Forward and Reverse
ReagentGoTaq® Green Master Mix Promega CorparationCatalog #M7123
Troubleshooting
Nf1 loxP site verification
15m
ReagentSpecific Primers Forward and Reverse
Primers for the Nf1 loxP1 site:
P1: 5’-CTTCAGACTGATTGTTGTACCTGA-3’
P4: 5’-TGATTCCCACTTTGTGGTTCTAAG-3’ 
P3: 5'-ACCTCTCTAGCCTCAGGAATGA-3'

Primers for the Nf1 loxP2 site:
LoxP2For: 5’-GCTTTAGCTTCTGGAAATGTGAA-3’ 
LoxP2Rev: 5’-GCGGGCTAAAATGGCAATGTCG-3’ 
gDNA preparation from NaOH lysate
  1. Cut a 2-3mm piece of tail/ear
  2. Add it to 300uL of lysis solution (NaOH 50mM)
  3. Heat for 15 min at 95C
  4. Centrifigationmax rpm, Room temperature, 00:15:00 , use max speed of your bench centrifuge


15m
PCR reaction
From a working solution of 10uM, use 1 uL of each primer (200nM each in a total volume of 20uL)
Reaction for loxP1: P1 + P2 + P3
Reaction for lopP2: LoxP2For + LoxP2Rev
Per reaction, mix 0.5uL of gDNA from the NaOH lysate (from step 2), 1 uL of each primer (from step 3.1), 10 uL ReagentGoTaq® Green Master Mix Promega CorparationCatalog #M7123 mix, and add water for a total volume of 20 uL

10m
Go to PCR cycle program
ABC
Temp.DurationCycles
94C5 min
94C15 sec3 to 5, 35x
58C30 sec
72C1 min
72C10 min
PCR temperatures and cycles

2h
Run PCR reaction products on a 2% ethidium bromide agarose gel
1h

Expected result
Expected PCR results:
 
a)         LoxP1 site:
480bp: WT (+) allele (P1-P3) in Nf1 +/+ or Nf1 f/+ mice, or loss of the loxP1 site in Nf1 f/f mice     
350bp: Presence of loxP1 (P1-P4) in Nf1f/f mice                                                                                               
 
b)        LoxP2 site:
699 bp: WT (+) allele in Nf1 +/+ or Nf1 f/+ mice, or loss of the loxP2 site in Nf1 f/f mice
829 bp: Presence of loxP2 in Nf1 f/f mice                                                                                                               

Protocol references
Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain, PMID:Y Zhu 1, M I Romero, P Ghosh, Z Ye, P Charnay, E J Rushing, J D Marth, L F Parada, PMID:11297510.