| First Author | Bennett MK | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 2 | Pages | 216-217 |
| Mgi Jnum | J:1275 | Mgi Id | MGI:49805 |
| Citation | Bennett MK, et al. (1992) A PCR based assay for the murine testicular feminisation mutation (Tfm). Mouse Genome 90(2):216-217 |
| abstractText | Full text of Mouse Genome contribution: A PCR based assay for the murine testicular feminisation mutation (Tfm). M.K.Bennett and H.M.Charlton; University of Oxford, Department of Human Anatomy, South Parks Road, Oxford OX1 3QX. Introduction Androgen insensitivity syndrome or testicular feminisation (Tfm) has been described in many animals such as rats (1), cattle (2) and man (3). The Tfm mutation only manifests itself in the male animal and causes the sexual development of an affected hemizygous Tfm XY male in utero to diverge from normal. The affected male exhibits a phenotype that has female external genitalia with paired intraabdominal testes and no Mullerian or Wolffian duct derivatives. The murine Tfm mutation was originally discovered among the offspring of a female homozygous for the X-linked genes mottled blotchy (Moblo) and tabby (Ta) (4). The genetic basis of the mutation has been reported recently and is a single base deletion in the gene coding for the androgen receptor. This deletion alters the open reading frame, such that a stop codon is introduced, producing a truncated RNA whose protein product is non-functional (5 and 6). Methods to identify heterozygous female carriers of the mutation are essential in order to breed significant numbers of the mutants and in the past the closely linked coat colour markers Moblo and Ta have been used to follow the inheritance of Tfm. The Tfm males produced from these crosses can be distinguished from their normal female litter-mates because they will be the only phenotypic females with the wildtype house-mouse coat colour of the background strain. However this means that identification of the mutants before coat colour differences are obvious necessitates dissection and observation of the internal genitalia. Any method that could dispense with the need to use coat colour markers for the identification of heterozygous female carriers and mutant males would not only aid a breeding program, but would allow developmental studies in Tfm mice to.be extended to the pre-natal, and neonatal period. With the knowledge that the Tfm mutation is a single base deletion a PCR protocol was designed that would produce a DNA fragment that spanned the region of the deletion. The differing sizes of fragment produced by the normal and mutant gene, differing by one base pair, can then be separated by a Polyacrylamide Urea sequencing gel. Materials and Methods A very small tail clipping (<0.5mm) was removed from the animal to be assayed and placed into a 0.75ml microfuge tube held on ice. Lysis of the tail tip to free genomic DNA was carried out as previously described (7). The lysis mix was then vortexed and centrifuged at 12,000g for 5 minutes, 10ul removed and placed into a fresh 0.75ml microfuge tube containing 10ul of the PCR reaction mix. A single PCR reaction contained 4pmoles of each of the 20mer unlabelled PCR primers TFM-4 and TFM-5 with less than 0.1pmole each of 32P end labelled TFM-4 and TFM-5 (1pmole=6.6ng of 20mer). Numbering of oligonucleotide placing as reference 5:- TFM-4 5'-AACTTTCCGCTGGCTCTGTC-3' (1123-1143 bp) TFM-5 5'-TACGGGCGTGTGGATGGGTA-3' (1188-1168 bp) 1.5mM MgCl2, dNTP's (Pharmacia) at 2mM each and 0.5IU of Taq DNA polymerase (Clontech) was also supplied per reaction. The sample was then overlayed with mineral oil (Sigma) and placed in a thermocycler (MJR thermocycler). The cycling times employed were as follows:- step 1 92 degrees C 4 minutes 45 seconds step 2 94 degrees C 15 seconds step 3 63 degrees C 15 seconds step 4 72 degrees C 30 seconds step 4 cycle 24 more times from step 2 step 5 end At the end of the PCR reaction the tubes were either frozen (-20 degrees C) or immediately size fractioned on a 6% Polyacrylamide Urea sequencing gel. The sequencing gel (Bio-rad sequi-gen sequencing cell 20x4(lcm) was set with a sharks tooth comb, to allow the analysis of 26 animals, and pre-run for at least 30 minutes at 1750 Volts to allow the gel to attain the correct running temperature of 55 degrees C. Loading buffer was added to the samples, 2ul removed and analysed on the gel. The sequencing gel was run for approximately 1 hour 45 minutes at 1650V. The urea was removed from the gel by soaking in 5% acetic acid 15% methanol for 15 minutes before the gel was dried (Bio-Rad Gel Drier) for 45 minutes at 80 degrees C. The gel was then placed in an autoradiography cassette with one rear intensifying screen and Amersham Hyperfilm-Beta-max. Exposure was for 12-24 hours at -70 degrees C. Results From figure 1 it can be seen that an unambiguous diagnoses of the genetic composition of the animals under analysis can be made. The normal animal producing a single band of 75bp, the carrier female producing two bands 75 and 74bp and the Tfm producing a single band of 74bp. Lane 1 Tfm, lane 2 normal male, lane 3 normal female and lane 4 carrier female (no difference noted with either Tfm++/+Ta+, Tfm++/++Moblo carrier females). Conclusion The genetic assay demonstrated in this paper will aid any breeding program for Tfm animals by allowing the user to dispense with coat colour markers. It will also allow the study of the developmental changes taking place pre-natally in Tfm animals and the recognition of new born Tfm animals which has been difficult. References 1. Bardin C.W., Bullock L., Schneidcr G,, Allison J.E. and Stanley AJ.: (1970) Science 167, 1136-1137. 2. Short R.V,: (1967) Annual Review of Physiology 29 373-400. 3. Morris J.M.: (1953) American Journal of Obstetrics and Gynaecology. 65, 1192-1195. 4. Lyon F.M, and Hawkes S.G,: (1971) Nature 227, 1217-1219. 5. Charest N.J., Zhou Z., Lubahn D.B., Olsen KL,, Wilson E.M. and French F.S.: (1991) Molecular Endocrinology 5(4), 573-581. 6. Gaspar M.L., Meo M., Bourgarel P., Guenet J.L., and Tosi M.: (1991) Proceedings on the National Academy of Science U.S.A. 88, 8606-8610. 7. Lang J.: (1991) Mouse Genome. 89(4), 857. |
