| First Author | Lu Y | Year | 1993 |
| Journal | Mouse Genome | Volume | 91 |
| Issue | 4 | Pages | 844-50 |
| Mgi Jnum | J:16289 | Mgi Id | MGI:64373 |
| Citation | Lu Y, et al. (1993) RAPD-PCR analysis of the C57BL/6FrSe-cho genome. Mouse Genome 91(4):844-50 |
| abstractText | Full text of Mouse Genome contribution: RAPD-PCR Analysis of the C57BL/6FrSe-cho Genome. Yujuan Lu, Bryan B. Wardell, Cory Teuscher, and Robert E. Seegmiller*. Departments of Zoology and Microbiology, Brigham Young University, Provo, UT 84602, U.S.A. Tel: 801-378-2303, Fax: 801-378-7499. *Author for correspondence. Abstract Chondrodysplasia (cho) is a well-described hereditary dwarfism in mice which serves as a model for human chondrodystrophy. The gene is an antosomal recessive mutation that has neither been mapped within the murine genome nor linked lo any known molecular markers. Since the technique of random amplification of polymorphic DNA (RAPD) has proven valuable in genome mapping, strain and parentage identification, and in the development of DNA-based markers linked to target genes, it was used in this study to analyze the C57BLJ6FrSe-cho genom While our efforts did not succeed in identifying markers linked to the cho mutation, they revealed the presence of hv RAPD markers, DOByu28 and DOByu29, segregating within the C57BLl6FrSe-cho subline. Both RAPD markers appear to be specific to this substrain in that they were not segregating within the C57BL16J. C57BL/6ByJ, C57BL/KsJ, C57BL/6N, C57BLl6St and C57BU6Td substrains. Screening of 142 C57BLl6FrSe-cho mice obtained from the colony identified 8 which expressed the C57BLl6J-type alleles for both RAPDS. Pedigree analysis suggested that the C57BLl6J-type alleles were introduced into the colony by prior out crossing to female C57BU6J mice. These results demonstrate that RAPD-PCR can be used in the genetic quality control of breeding programs within a closed colony, even at the substrain level. Introduction Chondrodysplasia (cho), which arose as a spontaneous mutation, is a well-described hereditary dwarfism in mice that serves as a model for human chondrodystrophy 1. The mutation occurred in the mid-sixties in the C57BL16Fr subline maintained at McGill University from 1945 to the early-70's. Subsequently, the mutation has been maintained as the C57BLl6FrSe-clw subline at Brigham Young University. The morphologic and histologic features of cho resemble that of a specific human dwarfism called diastrophic dysplasia 2.3. The cho mutation is an antosomal recessive disorder that results in abnormally thick collagen fibers in the extracellular matrix of hyaline cartilage 1A5.6. Biochemical studies of cho mice revealed the absence of a 43-Kd wllagenase-insensitive polypeptide in the cartilage 7, however, the relationship of this finding to the cho phenotype remains unknown. The cho mutation has neither been mapped within the m u ~geen ome nor linked to known molecular markers +. (+ Recently, using the MEV-linkage testing stock, the cho mutation has been mapped to chromosome 3 of the murine genome (unpublished). The PCR-based technique of random amplification of polymorphic DNA (RAPD), which employs single primers of arbitrary sequence, has been useful in genome mapping 8, 9, 10, 11, 1a2n d in the identification of DNA-based markers linked to target genes 10.13, The RAPD technique is principally a function of primer-template interaction and is based on the presence of appropriately spaced inverted repeats within PCR distance of each other 10. RAPD-PCR has the potential of identifying polymorphisms associated with mutations that arise as a result of single bp mismatches at one or both priming sites, deletion of pruning sites, insertions between priming sites resulting in their being beyond PCR distance, and insertion or deletions that alter the size of the amplified product 10-12. Such characteristics make RAPD-PCR genome analysis well suited for both mapping studies and the identification of DNA-based markers tightly linked to the cho gene. We report the RAPD-PCR analysis of the C57BU6FrSe-cho genome and demonstrate the utility of this technique in identifying substrain-specific polymorphisms which can be used in the genetic quality control of breeding programs within closed colonies. Materials and Methods Mice Livers from the C57BL/6FrSe-cho colony maintained at Brigham Young University, were obtained from three proven genotypes (15 cho/cho, 24 cho/+, and 2 +/+) and 101 mice of unknown genotype (cho/+ or +/+). Livers from the C57BL/6N and C57BL/6St mice were obtained from David Strong (NIH, Bethesda, MD), and the C57BL/6Td livers were obtained from the Trudeau Institute (Seranac Lake, N.Y.). Genomic DNA DNA was isolated from liver tissue according to the method of Bell et al. 14. Briefly, approximately 0.15 g of tissue was pulverized in liquid nitrogen, followed by cell lysis overnight at 37 degrees C in a buffer consisting of 75 mM NaCl, 24 mM EDTA, 0.5% SDS and 0.2 mg/ml proteinase K. The digest was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and the DNA was precipitated with 1/10 volume of 3 M sodium acetate and 1.5 volumes of isopropanol. The DNA was suspended in TE (10 mM Tris-HCl, pH 7.4; 1 mM EDTA), precipitated with 0.5 volume of 7.5 M ammonium acetate and 2 volumes of 100% ethanol at Ð20 degrees C, resuspended in TE buffer, diluted to 4 ng/ul concentration with TE (10 mM Tris-HCl, pH 7.4; 0.1 mM EDTA) and stored at 4 degrees C. DNAs from C57BL/6J, C57BL/KsJ, and C57BL/6ByJ mice were purchased from the Jackson Laboratory (Bar Harbor, Me). RAPD Analysis RAPD primers were purchased from Operon Technologies, Inc. (Alameda, Calif.). RAPD-PCR was performed as previously described 11, 15. Briefly, each 25 ul volume contained 10 mM Tris-HCL (pH 8.3), 50 mM KC1, 4.0 mM MgCl2, 2.0 ug/ml BSA, dATP, dCTP, dGTP, and dTTP (each at 0.2 mM; Pharmacia LKB, Piscataway, N.J.), 0.5 uM primer, 40.0 ng genomic DNA, and 0.5 units of DNA polymerase (AmpliTaq, Perkin-Elmer/Cetus, Norwalk, Conn.). PCR was performed on a Techne Model MW-1 Dri-Plate Cycler programmed for 45 cycles of 94 degrees C for 1.5 min, 33 degrees C for 2.0 min, and 72 degrees C for 2.5 min. Amplification products were resolved by electrophoresis for approximately 3 hours at 75 volts in 2.5% ME agarose gels (SeaKem, FMC, Rockland, Me) containing IX TAE buffer, and visualized with ethidium bromide. Results and Discussion For the initial genome screen, DNAs from C57BL/6J and C57BL/6FrSe-cho/cho mice were used. Approximately 2,200 discrete bands, ranging in size from 0.2-2.5 kb, were amplified from 487 primers. An average of 4.5 products with a mean size of 980 bp representing 4.4 kb of DNA were amplified per primer. This is equivalent to the examination of 10(4) bp of sequence polymorphism at primer binding sites (974 primer sites x 10 bases/primer) and 10(6) bases represented indirectly as RAPD fragment size variants (487 primers x 4.5 products/primer x 980 bp product). Of the 2,200 loci examined, two of them, DOByu28 (primer N-9) and DOByu29 (primer V-17), were found to be polymorphic. Segregation analysis employing 15 cho/cho, 24 cho/+, and 2 +/+ mice revealed that neither DOByu28 nor DOByu29 were linked to the cho mutation, i.e. both RAPD markers were present among animals regardless of the cho genotype. However, it was observed among these and 101 additional C57BL/6FrSe-cho subline mice of unknown genotype that DOByu28 and DOByu29 were concordant, suggesting that they are tightly linked to each other (Table 1). Table 1. Segregation of RAPDs in C57BL/6FrSe-cho mice. Locus: DOByu28 (present); RAPD primer sequence: TGCCGGCTTG (N-9); product size: 860 bp; Number of animals concordant/analyzed: cho/cho: 10/10; cho/+: 23/23; +/+: 2/2; cho/+ or +/+: 99/99; Total: 134/134. Locus: DOByu29 (present); RAPD primer sequence: ACCGGCTTGT (V-17); product size: 860 bp; Number of animals concordant/analyzed: cho/cho: 10/10; cho/+: 23/23; +/+: 2/2; cho/+ or +/+: 99/99; Total: 134/134. Locus: wild-type (absent); Number of animals concordant/analyzed: cho/cho: 5/5; cho/+: 1/1; +/+: 0/0; cho/+ or +/+: 2/2; Total: 8/8. Total Analyzed: cho/cho: 15; cho/+: 24; +/+: 2; cho/+ or +/+: 101; Total: 142. Analysis of other C57BL/6 substrains indicated that DOByu28 and DOByu29 were substrain specific in that neither were present in C57BL/6J, C57BL/6ByJ, C57BL/KsJ, C57BL/6N, C57BL/6St and C57BL/6Td mice (Fig. 1). The results indicate that at least one additional polymorphic locus unrelated to cho is segregating in the C57BL/6FrSe-cho subline. In the early 70's. the C57BL/6FrSe-cho substrain was separated from the C57BL/6Fr-cho colony maintained at McGill University (Fig. 2). Since that colony has been discontinued, it cannot be ascertained whether the two polymorphisms were fixed in the substrain prior to this point in time or subsequent to its establishment at Brigham Young University. Figure 1. (Legend). Amplification products obtained with genomic DNA from C57BL/6 substrains 6J, 6FrSe-cho, 6ByJ, KsJ, 6N, 6St, and 6Td, using RAPD primer V-17 (5' -> 3' sequence = ACCGGCTTGT). DOByu29 was present in C57BL/6FrSe-cho mice but not in other substrains of C57BL/6 mice. Similar results were obtained with RAPD primer N-9 (5' -> 3' sequence = TGCCGGCTTG) for the DOBYU28 locus. End lanes 1 and 16 are the 123-bp molecular weight markers. Figure 2. (Legend). Lineage of the substrains analyzed by the RAPD technique(16). Genomic screening of a total of 142 C57BL/6FrSe-cho mice revealed that eight expressed the C57BL/6J-type alleles for both DOByu28 and DOByu29 (Fig. 3). Through pedigree analysis (Fig. 4), it was possible to trace all eight mice to four C57BL/6J females (Jackson Lab, Bar Harbor) introduced in 1991 in an effort to expand the colony. The pedigree in Fig. 5 confirms the dominant inheritance pattern of the polymorphisms and further suggests a 1ow frequency for the C57BL/6J-type alleles in our colony. Of the 142 C57BL/6FrSe-cho mice tested, the two RAPDs were segregating in 134 mice, suggesting the frequency of these two markers in the C57BL/6FrSe-cho substrain is high. Figure 3. (Legend). Amplification of RAPD primer V-17 with genomic DNA from several C57BL/6FrSe-cho mice in comparison with C57BL/6J mice. Eight C57BL/6FrSe-cho mice showed the C57BL/6J-type allele, which proved consistent with C57BL/6J out-crossing with the C57BL/6FrSe-cho colony. The same results were obtained with RAPD primer N-9. End lanes 1 and 16 are the 123-bp molecular-weight markers. Our results indicate that RAPD-PCR can be used to identify DNA-based, substrain specific markers(12). Therefore, RAPD-PCR may be useful in the development of DNA-based markers for the genetic analysis of substrains of mice known to exhibit differential phenotypes for which molecular markers have not been identified. One classic example is the set of genetically controlled differential phenotypes observed among BALB/c substrains. These include, but are not limited to, susceptibility to plasmacytomagenesis (17) and experimental allergic orchitis and experimental allergic encephalomylitis (18). In addition, our results suggest that RAPD-PCR can be used to monitor evolutionary divergence as well as the genetic quality control of breeding programs within a closed colony even at the substrain level. Acknowledgments We thank Drs. J.L. Farmer, and S.R. Woodward for their helpful discussion and review of the manuscript, and Brad Seegmiller for drawing the pedigrees. This work was supported, in part, by National Institutes of Health grants HD-21926 and HD-27275, and a grant from the Professional Development Fund of Brigham Young University. Figure 4. (Legend). Pedigree of C57BL/6FrSe-cho mice showing the C57BL/6J-type alleles in eight mice, all of whose lineage traced back to four C57BL/6J female mice obtained from Jackson Laboratory and introduced into the colony in 1991. Stippled symbols represent DOByu28 and DOByu29 present (genotype +/+ or -/-); open symbols represent DOByu28 and DOByu29 absent (genotype +/+); question marked symbols represent untested animals; 6J represents C57BL/6J. Figure 5. (Legend). Representative pedigree of C57BL/6FrSe-cho mice in which both DOByu28 and DOByu29 were present in 134 mice tested, suggesting a high frequency of homozygosity for the polymorphism among the colony. Stippled symbols represent DOByu28 and DOByu29 present (genotype +/+ or -/-); question-marked symbols represent untested animals; 6J represents C57BL/6J, DOByu28 and DOByu 29 presumed absent (genotype presumed -/-). References 1. Seegmiller, R.E., Fraser, F.C. and Sheldon, H.: A new chondrodystrophic mutant in mice: electron microscopy of normal and abnormal chondrogenesis. J. Cell. Biol. 48: 580-593, 1971. 2. McKusick, V.A.: Mendelian inheritance in man, p. 477, The Johns Hopkins University Press, Baltimore, 1978. 3. 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