| First Author | Lehman DM | Year | 1997 |
| Journal | Mouse Genome | Volume | 95 |
| Issue | 3 | Pages | 689-691 |
| Mgi Jnum | J:43277 | Mgi Id | MGI:1097457 |
| Citation | Lehman DM (1997) RAPID TYPING OF LITTLE MOUSE MUTATION. Mouse Genome 95(3):689-691 |
| abstractText | Full text of Mouse Genome contribution: RAPID TYPING OF LITTLE MOUSE MUTATION. D.M. Lehman,1 K.E. Mayo,2 D.E. Hale,3 J.T. Cody,4 T.M. Poole,5 R.J. Leach1; 1Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78284-7762 USA. 2Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208 USA. 3Department of Pediatrics, Division of Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284-7762 USA. 4Clinical Investigations, Wilford Hall Medical Center, Lackland AFB, Texas 78236 USA. 5McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 USA. Introduction The little mouse mutant (lit) exhibits postnatal growth retardation and hypomyelination resulting from insufficient levels of growth hormone and insulin-like growth factor 1 (IGF-1) [3, 4]. The deficiency of these factors is due to a recessively inherited missense mutation in which a conserved aspartic acid (GAT) at position 60 is mutated to glycine (GGT) in the growth hormone releasing hormone receptor (Ghrhr) gene. This mutation has been shown to disrupt normal signalling by the receptor [1]. Pituitary growth hormone levels of homozygous lit mice are reduced to 6 to 8% of levels in heterozygous littermates [5], but early postnatal treatment with growth hormone ameliorates the developmental defects [6]. The lit mouse mutant is therefore a useful model for human isolated growth hormone deficiency type I [3]. Moreover, a nonsense mutation in the human homolog (GHRHR) has recently been identified in a family with growth hormone deficiency [7]. This mutation creates a stop codon at amino acid position 72 near the site of the mouse lit mutation and predicts a truncated, dysfunctional protein. Evidence for the inactivation of the human homolog in the etiology of some growth hormone deficiencies further emphasizes the benefit the lit mouse model has for studies of this disorder. Generally, homozygous C57BL/6J lit mice can be distinguished from their wild-type littermates by size. Homozygous lit mice grow only to about two-thirds the size of wild-type mice of the same strain [3]. However, if this mutant is crossed with a different strain or with another mutant, selection by phenotype becomes difficult. Additionally, heterozygotes and wild-type mice have indistinguishable phenotypes. This quick PCR-based method is simple and clearly identifies heterozygotes. Moreover, genotyping by this method facilitates the study of early lit embryos for various developmental studies, Materials and Methods Method of sequence analysis: DNA encompassing the second intron of Ghrhr and the little point mutation was obtained by PCR amplification of genomic DNA from C57BL/6J +/+, +/lit, and lit/lit mice as previously described [1, 2]. The primers used were 5Ã- A C T G G G T C A C C T C C A C C T A G A A T G - 3' (MutC) and 5' - CCTGTGTCTGAGCCGAAGTGAGAG-3' (MutD). Each 20 ul reaction consisted of 200 ng genomic DNA, 400 ng each primer, 1.5 mM Mg++, and 0.2 mM dNTPs. Amplification was carried out for 50 cycles using an annealing temperature of 65 degrees C with additional Taq polymerase (Perkin Elmer-Cetus) added after 35 cycles. PCR fragments were cloned into pGEM3Z(+/+), pGEM7Z(+/lit), or pGEM4Z(lit/lit) and their sequence determined using Sequenase (US Biochemicals). (Fig. 1A) Allele detection: Restriction analysis of the above PCR products was used to detect the presence of the transition mutation in the third exon of Ghrhr. Briefly, 10ul of each 20ul PCR reaction were double digested with Fokl and NlaIII (New England Biolabs) in NEB4 buffer at 37 degrees C for 2 hours. Following electrophoresis of the digest on a 9% polyacrylamide gel, which was run at 120V for 4 hours, differential bands were detected by staining the gel with ethidium bromide at a concentration of 0.5 ug/ml. (Fig. 1B) Fig. 1 (Legend) (A) Shows the region of Ghrhr that was PCR amplified as described in the text. Arrows denote PCR primers; star marks nucleotide at which lit mutation occurs; solid boxes are Fokl or NlaIII recognition sites; stippled box is intron 2 sequence. (B) Polyacrylamide gel showing differential bands from wild-type and lit Ghrhr. Results and Discussion We have developed a method for genotyping the little mutation in mice from genomic tail DNA. Using this technique, we have been able to distinguish true little littermates from the runt of the litter at an early age. Also, heterozygotes and wild type mice can now be differentiated. Previously identified homologs: Human homolog is growth hormone releasing hormone receptor (GHRHR) mapped to 7p14. Database deposit information: GenBank accession numbers L07379 and U81979 (intron 2). Acknowledgements: This work was supported in part by a grant from the Genentech Foundation for Growth and Development (DML, DEH, RJL) as well as National Institutes of Health grant PK48071 (KEM) and USAF Surgeon General's Office grant SG094-187 (JTC). Support was also received by National Cancer Institute grants CA22484 and CA07175 (TMP). The authors thank Wes Beamer (Jackson Laboratory) for his helpful assistance and Matt Mailman for his technical support. Additionally, the authors wholeheartedly thank the animal care staff at Wilford Hall. RJL is the corresponding author. References 1. Godfrey, P., Rahal, J.O., Beamer, W.G., Copeland, N.G,, Jenkins, N.A., Mayo, K.E. (1993). Nature Genet. 4, 227-232. 2. Lin, S-C., Lin, C.R., Gukovsky, I., Lusis, A.J., Sawchenko, P.E., Rosenfeld, M.G. (1993). Nature 364, 208-213. 3. Eicher, E.M., Beamer, W.G. (1976). J. Hered. 67, 87-91. 4. Donahue, L.R., Beamer, W.G. (1993). J. Endocrinol. 136, 91-104. 5. Cheng, T.C., Beamer, W.G., Phillips III, J.A., Bartke, A., Mallonee, R.L., Dowling, C. (1983). Endocrinol. 113(5), 1669-1678. 6. Noguchi, T. (1991). Comp. Biochem. Physiol. 98C(1), 239-248. 7. Wajnrajch, M.P., Gertner, J.M., Harbison, M.D., Chua Jr., S.C., Leibel, R.L. (1996). Nature Genet. 88-90. |
