| First Author | Estes SS | Year | 1994 |
| Journal | Mouse Genome | Volume | 92 |
| Issue | 1 | Pages | 130-32 |
| Mgi Jnum | J:17344 | Mgi Id | MGI:65391 |
| Citation | Estes SS, et al. (1994) RAPD-PCR analysis of C57BL/10 H-2 congenic strains of mice. Mouse Genome 92(1):130-32 |
| abstractText | Full text of Mouse Genome contribution: RAPD-PCR Analysis of C57BL/10 H-2 Congenic Strains of Mice S.S. Estes, B.B. Wardell, S.R. Woodward and C. Teuscher*; Department of Microbiology, Brigham Young Univeristy, Provo, UT 84602; Tel: 801-378-5712, Fax: 801- 378-7499 *Author for correspondence. Abstract Congenic strains of mice are used to assist in linkage analysis and to compare the effects of genes without background interference. The H-2 congenic strains C57BL/10J (B10), B10.BR/SgSnJ and B10.D2/nSnJ were developed to study H-2 and its characteristics. In this study, these strains were used as prototypes to assess the capability of random amplification of polymorphic DNA (RAPD) to identify molecular-based markers linked to donor derived segments on the inbred partner background. Three hundred sixty-five primers were used to identify polymorphisms distinguishing B10 and C57BR/cdJ and B10 and DBA/2J. Fifty-eight of the primers identified RAPD markers distinguishing B10 and C57BR/cdJ while 75 primers differentiated B10 and DBA/2J. The same primers were used to screen B10, B10.BR/SgSnJ and B10.D2/nSnJ mice. Three of the donor strain RAPDs segregated among the congenic lines. Recombinant inbred lines (RIL) were used to map all three RAPD markers. D17Byu2 and D17Byu3 linked to D17Mit10 which maps 10 cM distal of H-2; and D1Byu10 which distinguishes B10 and B10.D2/nSnJ linked to Aox-1, Len-1 and Len-2 on Chr 1. These results support the utility of RAPD-PCR in identifying DNA-based markers linked to the gene(s) of interest in congenic strains as well as in identifying unlinked contaminating donor strain loci. Introduction Theoretically, congenic strains possess identical genomes with the exception of a differential donor strain segment encoding the gene(s) of interest (1). Over 500 congenic strains of mice exist, the majority of which involve either immunologically important loci (2), mutant genes or biochemical loci (3). Construction of congenic lines is generally initiated by mating a donor strain expressing a locus of interest with the prospective inbred partner. In successive generations, a carrier of the differential segment is backcrossed to the inbred partner. The line is made homozygous for the differential locus by full sibling mating after ten to twelve backcrossings (4, 5). The precision of studies utilizing congenic strains is, in part, dependent upon how thoroughly background donor loci unlinked to the locus of interest are eliminated from the inbred partner genome and how well the differential segment has been characterized (6, 7). Current molecular techniques for characterizing these donor derived segments include restriction fragment length polymorphisms (8), variable number tandem repeats (9), and simple sequence length polymorphisms or microsatellites (10). However, these techniques require cloned probes or pre-existing sequence data and, for the most part, target single loci for analysis. In contrast, RAPD-PCR allows for the simultaneous screening of multiple loci across a genome while using a single primer of arbitrary sequence (11-13). In this study we demonstrate the utility of RAPD-PCR in identifying DNA-based markers linked to both the differential segment of interest as well as contaminating donor strain loci. Materials and Methods DNA was isolated from liver tissue of the various strains studied (14). Briefly, 0.5 g of tissue, maintained in liquid nitrogen, was pulverized with a mortar and pestle. The cells were lysed and deproteinized with SDS and proteinase-K followed by phenol/ chloroform/isoamyl alcohol extraction(s). The DNA was then precipitated in sodium acetate with isopropanol, suspended in TE (10 mM Tris-HCl, pH 7.4. 1 mM EDTA), reprecipitated with ammonium acetate and ethanol, and resuspended in TE. Working aliquots of each DNA sample were prepared by bringing them to a final concentration of 4 ng/ul in TE (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA). DNA for the BXD RILs was purchased from The Jackson Laboratory (Bar Harbor, ME). DNA for the AXB and BXA RILs was a gift from Dr. Beverly Paigen. The Jackson Laboratory (Bar Harbor, ME). RAPD primers were purchased from Operon Technologies, Inc. (Alameda, CA). RAPD-PCR was carried out as previously described (12,13). Briefly, the reactions were performed in a 25-ul reaction volume containing 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), 0.5 uM primer, 40.0 ng genomic DNA, and 0.5 units of DNA polymerase. Amplification 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. Reaction products were resolved by electrophoresis at 75 V for 4 h in 2.5% ME agarose gels containing 1X TAE and visualized with ethidium bromide. Results and Discussion For the initial genome screen, DNAs from B10, DBA/2J and C57BR/cdJ mice were used. Approximately 1,600 discrete bands, ranging in size from 0.1-3.0 kb, were amplified with 365 primers. On average, 5.8 products with a mean size of 980 bp representing approximately 5.6 kb of DNA were amplified per primer. Of the 365 primers used, 58 exhibited RAPDs which distinguished B10 and C57BR/cdJ while 75 resulted in polymorphisms between B10 and DBA/2J. Subsequent genome screens of B10 and B10.BR/SgSnJ with the 58 distinguishing primers revealed one 1,170 bp C57BR/cdJ RAPD segregating in B10.BR/SgSnJ (Operon Primer G12, 5'-CAGCTCACGA-3'). Screening B10 and B10.D2/nSnJ with the 75 informative primers resulted in the identification of two DBA/2J RAPDs, one of 680 bp (Operon Primer C20, 5'-ACTTCGCCAC-3') and one of 2,100 bp (Operon Primer Il5, 5'-TCATCCGAGG-3'), segregating in B10.D2/nSnJ mice (Figure 1). Figure 1. (Legend). Amplification products obtained with genomic DNA from C57BL/10J, B10.BR/SgSnJ, C57BR/cdJ, B10.D2/nSnJ, and DBA/2J using Operon primers C20 (Dl7Byu2), G12 (Dl7Byu3) and Il5 (DlByu10). First lane of each set is a 123 bp molecular weight ladder. The three RAPDs were mapped using RILs whose parental strains exhibited the same fragment size variants as thc congenic pairs. The 1,170 bp RAPD generated with primer G12 was found to distinguish C57BL/6J and A/J thereby allowing for the use of the AXB and BXA RILs. Similarly, the 2,100 bp RAPD amplified with primer Il5 was also polymorphic between C57BL/6J and A/J. In addition, it was polymorphic between C57BL/6J and DBA/2J as was the 680 bp RAPD generated with primer C20. The strain distribution patterns (SDP) for each are summarized below in Table 1. Table 1. SDPs of RAPDs segregating in B10 H-2 congenic mice. Primer C20 (D17Byu2); BXD 1: D; 2: D; 5: D; 6: D; 8: B; 9: B; 11: D; 12: B; 13: D; 14: B; 15: B; 16: D; 18: D; 19: B; 20: D; 21: B; 22: D; 23: B; 24: D; 25: D; 27: D; 28: D; 29: B; 30: D; 31: D; 32: D. Primer G12 (D17Byu3); AXB 1: A; 2: A; 3: A; 4: B; 5: A; 6: B; 7: A; 8: A; 9: B; 10: B; 11: B; 12: B; 13: U; 14: B; 15: B; 17: A; 18: B; 19: B; 20: B; 21: A; 23: B; 24: A. BXA 1: B; 2: B; 4: A; 7: B; 8: B; 9: A; 11: B; 12: A; 13: B; 14: B; 16: B; 17: B; 18: A; 20: B; 22: B; 23: A; 24: B; 25: A; 26: B. Primer Il5 (D1Byu10); BXD 1: D; 2: B; 5: D; 6: D; 8: D; 9: B; 11: B; 12: B; 13: B; 14: B; 15: D; 16: D; 18: D; 19: D; 20: D; 21: D; 22: B; 23: B; 24: B; 25: D; 27: D; 28: U; 29: D; 30: D; 31: B; 32: B. AXB 1: A; 2: A; 3: B; 4: A; 5: A; 6: B; 7: A; 8: A; 9: B; 10: A; 11: B; 12: B; 13: B; 14: B; 15: B; 17: B; 18: A; 19: A; 20: A; 21: A; 23: B; 24: B. BXA 1: A; 2: B; 4: A; 7: A; 8: B; 9: B; 11: A; 12: A; 13: A; 14: A; 16: B; 17: B; 18: B; 20: A; 22: A; 23: B; 24: B; 25: B; 26: A. Linkage analysis employing MAP MANAGER (15) resulted in linkage of the RAPDs defined by primers C20 and G12 with D17 Mit10 at approximately 10 cM from H-2, well within the differential segment as previously defined (8). Accordingly, they have been designated D17Byu2 and D17Byu3 respectively. In contrast, the RAPD defined by primer 115 was linked to Aox-1, Len-1 and Len-2. Linkage analyses was performed using a conventional format, minimizing for double recombinants, and were significant at or below the 99% confidence level. Acknowledgments This work was supported by NIH grants HD-21926 and HD-27275, and a grant from the Professional Development Fund of Brigham Young University. References 1. Snell, G.D. (1948) J. Genetics 49:783-788. 2. Klein, J.: Congenic and segregating inbred strains. Immunologically important loci. In M.F. Lyon and A.G. Searle (eds.): Genetic Variants and Strains of the Laboratory Mouse, 2nd edn. pp. 797-825, Oxford University Press, Oxford, 1989. 3. Lane, P.W. and Lyon, M.F.: Congenic and segregating inbred strains. Mutant genes and biochemical loci. In M.F. Lyon and A.G. Searle (eds.): Genetic Variants and Strains of the Laboratory Mouse, 2nd edn. pp. 825-842, Oxford University Press, Oxford, 1989. 4. Haldane, J.B.S. (1948) J. Genetics 49:104-108. 5. Green, E.L.: Genetics and Probability in Animal Breeding Experiments. Macmillan Press, London, 1981. 6. Klein, D., et al. (1982) Immunogenetics 16:319-328. 7. Vincek, V., et al. (1990) Immunogenetics 31:45-51. 8. Botstein, D., et al. (1980) Am. J. Hum. Genet. 32:314-331. 9. Jeffreys, A.J., Neuman, R., and Wilson, V. (1990) Cell 60:473-485. 10. Lilt, M. and Luty, J.A. (1989) Am. J. Hum. Genet. 44:397-401. 11. Welsh, J. and McClelIand, M. (1990) Nucl. Acids Res. 18:7213-7218. 12. Woodward, S.R., Sudweeks, J., and Teuscher, C. (1992) Mamm. Genome 3:73-78. 13. Wardell, B.B., et al. (1992) Mamm. Genome 4:109-112. 14. Bell, B., Karam, J., and Rutler, W. (1981) Proc. Natn. Acad. Sci. USA 78:5759-5763. 15. Manly, K.F. and Elliot, R.W. (1991) Mamm. Genome 1:123-126. |
