| First Author | Lund L | Year | 1997 |
| Journal | Mouse Genome | Volume | 95 |
| Issue | 4 | Pages | 889-91 |
| Mgi Jnum | J:45473 | Mgi Id | MGI:1195494 |
| Citation | Lund L, et al. (1997) PCR typing of obese mice. Mouse Genome 95(4):889-91 |
| abstractText | Full text of Mouse Genome contribution: PCR typing of obese mice. Lene Lund1, Frederik Dagnaes-Hansen2 and Klaus Kristensen1, 3. 1Bomholtgard Breeding and Research Centre, DK-8680 Ry. 2Dept. of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C. 3TO whom correspondence should be addressed. Introduction The mouse Lepob mutation is a single base nonsense mutation in codon 105 of the gene encoding leptin (1, 2). In homozygous mutants the protein leptin, predominantly expressed in adipose tissue of normal mice, is missing (3). This results in profound obesity and type II diabetes. (4, 5). Obese mice are used as a model of obesity in humans. In Lepob/Lepob mice phenotypic expression of obesity and other symptoms of type II diabetes are first recognizable at about 4 weeks of age. Lepob/+ mice are phenotypically indistinguishable from wild-type mice (6). Lepob/Lepob females are sterile; only on rare occasions fertile males can be found (5). Thus mating has to be done with heterozygotes. It has however until now only been possible to identify heterozygous breeders, by a previous testmating with mates that were known to carry the obese allele. This all mounts to the fact that breeding of obese mice has been a complicated, time consuming and tedious matter. A fast typing and screening method has been missing due to the fact that only a single base substitution distinguishes obese carriers from wild-type mice. PCR is frequently used to assay for polymorphisms and pathogenic mutations. As the base substitution in codon 105 of the leptin gene generates a Dde I restriction site, a PCR protocol can be used to type this restriction site polymorphisms. In this protocol the polymorphism is amplified from genomic DNA by use of primers flanking the putative Dde I site. Then the amplified DNA is cut with the restriction enzyme and the fragments are separated by agarose gel electrophoresis (7, 8). A more simple strategy for mutation detection using PCR is the amplification refractory mutation system (ARMS) (9), which is based on the fact, that the DNA synthesis step in a PCR reaction is crucially dependent on correct base pairing at the 3' end. We here present a PCR protocol for rapid typing and screening of the obese mutation based on ARMS. The method is used routinely to screen our colonies for new breeders. Materials and Methods. Strains: Typing has been performed on the strains C57BL/6JBom-ob and Umea/Bom-ob. C57BL/6Jbom-ob is congenic with C57BL/6J and is maintained by close inbreeding and backcrossing. Umea/Bom-ob is a non-inbred strain (10). DNA isolation: DNA is extracted from tail biopsies using a standard protocol (11). After lysis of the biopsies with proteinase K, the DNA is precipitated with isopropanol and redissolved in H2O at a concentration of 1 ug/ul. Primers: For detection of the Lepob allele the OB-F/OB-MUT primer set is used to amplify a 155 bp fragment: OB-F: 5'- TTG GAC TTC ATT CCT GGG CTT C - 3' / OB-MUT: 5'- CAG CAG ATG GAG GAG GTC TCA - 3'. Detection of the Lep wildtype allele is done by amplifying a fragment of similar size by replacement of the OB-MUT primer with the OB-WT primer in the primer set: OB-WT: 5'- CAG CAG ATG GAG GAG GTC TCG - 3'. As an endogenous control of the reaction, the respective OB primer sets are co-amplified with the APC-F/APC-B primer set: APC-F: 5'- CCG GAG TAA GCA GAG ACA CAA GC -3' / APC-B: 5'- CCA ATA CCT CGC TCT CTC TCC A - 3'. These primers give rise to a 223 bp fragment. PCR-mixture: A total reaction volume of 100 ul is used: 2u1 genomic DNA (1 ug/ul), 1 ul of each of the four primers (DNA Technology; each 1 ug/ul), 10 ul 10 x PCR buffer (Perkin Elmer Cetus; 500 mM KC1, 100 mM Tris-HCl, pH 8.3), 12 ul MgCl2 (25 mM), 1 ul dNTP mix (Stratagene; each 25 mM), 0.5 ul Taq Polymerase (Perkin Elmer Cetus; Ampli Taq Gold 5 units/ul) and 70.5 ul ddH2O. Reaction conditions: PCR is performed using the Perkin Elmer 9600 temperature cycling system. All reactions are run in parallel with positive and negative DNA-controls as well as blank reaction- controls. The Taq Polymerase is activated for 10 minutes at 94 degrees C. This is followed by 30 cycles at 95 degrees C for 15 seconds and 67 degrees C for 30 seconds. No specific extension step is included in the first 30 cycles. Then a final extension of partial PCR-products are performed at 72 degrees C for 10 minutes. Note, that no specific extension step is included in the first 30 cycles. After completion of cycling, samples are cooled to 4 degrees C and 20 ul of the reaction volume are removed and loaded on a 1.5 % agarose gel. The gel is run at 65 V in TEA-buffer for 3 hours. The amplified fragments are visualized by staining with EtBr and UV transillumination of the gel. Results and Discussion An example of the obese PCR-typing reactions is shown in figure 1. Genomic DNA from three mice of known 'obese' genotype has been typed for the Lep allele using the OB-F/ OB-WT primer set (upper panel); and for the Lepob allele using the OB-F/OB-MUT primer set (lower panel). The amplified 155 bp fragments resulting from either primer set correspond to positions 234-389 in mouse Lep- cDNA (1, Mouse Genome Database). The OB-WT and OB-MUT primers only differ in their utmost 3' bases, which are complementary to position 369 in Lep-cDNA. This base is a C in the wild type allele, and a T in the obese allele. Due to this diminutive difference between the two primers and the delicacy of the resulting differential annealing, we find it important to run both negative and positive DNA controls when typing unknown samples. In addition we find it critical to follow the optimized reaction condition given above. Figure 1: (Legend). PCR-typing of the three different 'obese' genotypes. In both panels, co-amplification of the APC-F/APC-B primers set is used as an endogenous control of successful DNA preparation and reaction conditions. This primer set amplifies a fragment of 223 bp corresponding to positions 2348-2571 in Apc-cDNA (12, Mouse Genome Database). This sequence is identical in both obese and non-obese mice. Taken together, these typing reactions identify the three different genotypes unambiguously. We use the protocol routinely to select new breeders and heterozygous test animals from our colonies of C57BL/6J-ob and Umea-ob mice. The method works equally well on both strains. In the screening for new breeders, the protocol is simplified by the fact that only typing of Lepob/+ or +/+ is relevant. In this situation we have found it sufficient just to run the OB-F/OB-MUT reaction. Provided the endogenous APC-control is used, the information provided by this single reaction is sufficient to identify the obese genotype. We have until now typed 402 pups of 1ean phenotype from matings between heterozygous parents. Of these, 284 have been typed as Lepob/+. This number is in good accordance with an expected ratio of 2/3 heterozygotes among the lean phenotypes. When mated to previously test mated heterozygotes 96% of the typed heterozygotes became parents to phenotypically obese mice in the first litter. Thus the specifity of the typing-assay is close to l00%, but probably can be improved by experience and further optimization. Acknowledgements Ms. Jonna Jensen is greatly acknowledged for experienced husbandry, tail clippings and test matings. Mr. Christian Holmberg, Dept. of Molecular Cell Biology, University of Copenhagen, is greatly acknowledged for initial aid in design of primers. References (1) Zhang, Y., Proenca, R., Maffai, M., Barone, M., Leopold, L. and Friedman, J.M. (1994) Nature 372: 425-432. (2) Friedman, J.M., Leibel, R.L., Siegel, D.S., Walsh, J. and Bahary, N. (1991) Genomics 11: 1054-1062. (3) MacDougald, O.A., Hwang, C.S., Fan, H. and Lane, M.D. (1995) Proc. Natl. Acad. Sci. USA 92: 9034-9037. (4) Coleman, D.L. (1978) Diabetologia 14: 141-148. (5) Charlton, H.M. (1984) Q. J. Exp. Physiol. 69: 665-676. (6) Coleman, D.L. (1979) Science 203: 663-665. (7) Krogh-Pedersen, H. (1996) Ph.D Thesis, Aarhus University. (8) Erickson, J.C., Hollopeter, G. and Palmiter, R.D. (1996) Science 274: 1704-1707. (9) Newton, C.R., Graham, A., Heptinstall, L.E., Summers, C., Kalsheker, N., Smith, J.C. and Markham, A.F. (1989) Nucleic Acids Research 17: 1503-2516. (10) Westman, S. (1968) Diabetologia 4: 141-148. (11) Laird, P.W. (1991) Nucleic Acids Research 19: 4293-4296. (12) Su, L.-K., Kinzler, K.W., Gould, K.A. and Dove, W.F. (1992) Science 256: 668-670. |
