Genotyping protocol for Gy mice (August, 2001)
The Jackson Laboratory utilizes visible phenotype to genotype animals from the B6EiC3Sn-a/A-Gy (Stock Number 000503) colony. Animals carrying the allele display a circling behavior whereas animals do not.
The following reference provides PCR assay information that may be used to genotype animals. We do not have experience using the protocols, but offer them as potential alternatives to phenotyping methods.
Strom TM, Francis F, Lorenz B, Boddrich A, Econs MJ, Lehrach H, Meitinger T. 1997. Pex gene deletions in Gy and Hyp mice provide mouse models for X-linked hypophosphatemia. Hum Mol Genet 6:165-171.
The Jackson Laboratory used to use visible phenotype to genotype animals from the B6.Cg-PhexHyp/J (stock number 000528) colony. Animals carrying the PhexHyp allele have reduced body size with shortened hind limbs and tail whereas animals with the Phex wild type allele do not.
The Jackson Laboratory utilizes a combination of breeding scheme and visible phenotype to genotype animals from its B6.Cg-Lepob (stock number 000632) and BKS.Cg-Lepob/+ (stock number 000696) colonies.
A cross between B6129PF1-Aw-J/Aw females transplanted with Lepob/Lepob homozygous ovaries and C57BL/6J males produces heterozygous offspring. Homozygous (Lepob/Lepob) progeny from matings of these Lepob/Lep+ heterozygotes are identified phenotypically by their obesity as early as ~4 weeks of age.
Lep ob genotyping protocols:
Lac-Z detection in tail biopsies
0.2g KH2 PO4
to 1 liter with DDI H2O
made in 1X phosphate buffered saline
5mM potassium ferricyanide
5mM potassium ferrocyanide
1 mg/ml X-gal (in dimethyl sulfoxide) final concentration
0.1M Hepes (pH 7.4)
made in 1X SpM
Murti, JR and Schimenti, JC. "Microwave-Accelerated Fixation and Lac-Z Activity Staining of Testicular Cells in Transgenic Mice". Analytical Biochemistry :198, pp. 92-96 (1991).
Mutant = A
Wild type = G
Genotyping protocol provided by the donating investigator of strain BALB/cJ-Trfhpx/J has provided this genotyping protocol as a courtesy. The Jackson Laboratory has not used this protocol.
F 5' tcaagtccaccaccaaggacc 3'
R 5' ctgggtttctctgcgacctctc 3'
The amounts of primer solution correspond to ~20 pmol of each primer. Genomic DNA was prepared using the Qiagen kit and eluted in 200 ul of water.
Clean amplified fragment using phenol/chloroform/iso-amyl alcohol mix (25:24:1). Resuspend in 10 ul of miliQ water.
The enzyme to be used is Hyp CH4 IV (NEB), an isoschizomer of Mae II. This enzyme works in low salt concentrations and acts poorly on samples taken out straight from the PCR tube. Run samples in a 1.5% agarose gel.
1-5 µl DNA 2.5 µl 10x Perkin Elmer buffer, 1.5 mM MgCl2 (final) 2 µl 2.5 mM dNTP mixture (2.5 mM each dNTP, 200 µM final) 0.5 µl 20 µM forward primer (0.4 µM final) 0.5 µl 20 µM reverse primer (0.4 µM final) 0.1 µl Taq DNA polymerase (can decrease to 0.05 µl) dH2O to 25 µl
(Adapted from Higuchi, R. (1989) Amplifications 2: 1-3)
Method that quantifies DNA sequences by normalizing to a reference gene, or to the amount of DNA in the reaction. QPCR is the easiest way to differentiate homozygous (Tg/Tg) from hemizygous (Tg/0) transgenic mice or to determine transgene copy number.
For more information:
Method that uses the melting point of double-stranded PCR products to determine the size of the DNA fragments amplified for transgenes and knock-ins (eliminating the need for agarose gels). This requires a fluorescent dye (typically SYBR green) and special thermocycler (e.g. LightCycler ® 480 from Roche). Melting curve analysis protocols provided on JAX ® Mice datasheets can be used as a starting point for standard PCR, by eliminating the SYBR green and optimizing the protocol for use in a standard thermocycler.
Sequencing method that uses a chemiluminescent enzyme to detect specific nucleotide incorporation into DNA to genotype knockout and spontaneous mutants. For more information: