Strain Protocols

Strain Information
Strain Code: 01XM3
Strain Name: 129S4-Trp53tm3Tyj/Nci
Gene Symbol: Trp53
Protocol Information
Allele: Trp53<tm3Tyj>
Protocol Number: 1
Introductory Comment: These mice express a point mutation in the p53 gene that can be activated by Cre-mediated recombination. Line 129S4-Trp53(tm2Tyj) - AKA P53 LSL R172H (01XM2)- contains a point mutation in Exon 5, converting an Arg into a His. Line 129S4-Trp53(tm3Tyj) - AKA p53 LSL R270H (01XM3) - contains a point mutation in Exon 8, converting an Arg into a His. These 2 lines contain LoxP sites and a transcriptional / translational STOP sequence, making it functionally equivalent to a null mutation. The genotyping strategy detects the WT with T037 and T035, which amplify intron 1, and the Mut with T036 and T035, which amplifies the LSL element. These primers do not distinguish R172H (01XM2) from R270H (01XM3). Sequencing may be needed to confirm the mutation.

Primers

Y582 : 5'-gtc tgg aat tcc gca agc ta-3'
T035 : 5'-ctt gga gac ata gcc aca ctg-3'
Y583 : 5'-aga gcc aaa gat aaa ggg taa aag-3'
Diagram showing the primer relataionships for Strain 01XM3.  These mice express a point mutation in the p53 gene that can be activated by Cre-mediated recombination.  Line 129S4-Trp53(tm2Tyj) - AKA P53 LSL R172H (01XM2)- contains a point mutation in Exon 5, converting an Arg into a His.  Line 129S4-Trp53(tm3Tyj) - AKA p53 LSL R270H (01XM3) - contains a point mutation in Exon 8, converting an Arg into a His.  These 2 lines contain LoxP sites and a transcriptional / translational STOP sequence, making it functionally equivalent to a null mutation.  The genotyping strategy detects the WT with T037 and T035, which amplify intron 1, and the Mut with T036 and T035, which amplifies the LSL element.  These primers do not distinguish R172H (01XM2) from R270H (01XM3).  Sequencing may be needed to confirm the mutation.

Product Sizes

Primer Combination Product Size
Y582 / T035 299 bp    Mut (LSL Element)
Y583 / T035 366 bp    WT

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0 mM
2.5mM dNTP Mix 0.2 mM
100 µMY582 0.5 µM
100 µMT035 1.2 µM
100 µMY583 0.5 µM
5U/┬Ál Taq Pol 0.015 U/µl
Use 50 - 100 ng of DNA per reaction

Cycling Conditions

(Time in minutes:seconds)
Temperature Time Label
94ºC 3:00
94ºC 1:00
60ºC 2:00 35 Cycles
72ºC 1:00
72ºC 3:00
4ºC HOLD

Disclaimer: PCR conditions were optimized using QIAGEN-purified mouse tail DNA and a PTC-200 thermocycler (MJ Research). Optimal conditions may vary with DNA quality or equipment used.