| First Author | Winton DJ | Year | 1992 |
| Journal | Mouse Genome | Volume | 90 |
| Issue | 4 | Pages | 690-92 |
| Mgi Jnum | J:3490 | Mgi Id | MGI:52003 |
| Citation | Winton DJ, et al. (1992) Derivation of a Dlb-1-a homozygous mouse congenic to C57BL/6J. Mouse Genome 90(4):690-92 |
| abstractText | Full text of Mouse Genome contribution: DERIVATION OF A Dlb-1a HOMOZYGOUS MOUSE CONGENIC TO C57BL/6J. D.J. Winton*, B. Gwynne + and B.A.J. Ponder*; *CRC Human Cancer Genetics Research Group, University of Cambridge, Tennis Court Road, Cambridge CB2 1 QP; +Institute of Animal Physiology & Genetic Research, Babraham Hall, Cambridge CB2 4AT. Introduction The Dlb-1 locus (chromosome 11) determines a unique reciprocal pattern of expression of a binding site for lectins recognising terminal nonreducing N-acetyl galactosamine residues, e.g. Dolichos biflorus agglutinin (DBA)(1, 2). We have derived a mouse congenic to C57BL/6J in which the usual Dlb-1b allele is replaced by Dlb-1a. Dlb-1b homozygote strains (e.g. C57BL/6J) express the lectin binding site on intestinal epithelium but not vascular endothelium (G+E- phenotype) while Dlb-1a homozygotes (e. g. SWR) display the converse pattern of expression (G-E+) (3, 4). A conjugate of peroxidase to DBA (DBA-Px) permits the dimorphism to be demonstrated histologically and this has allowed it to be used as a clonal marker to investigate the biology of gut epithelium (5, 6). Two different categories of experiment have been performed : (1) analysis of tissue mosaicism in aggregation chimaeras between Dlb-1b and Dlb-1a homozygote strains; (2) induction of DBA-Px non-staining clones in Dlb-1b /Dlb-1a heterozygotes which arise by mutation of the single b allele in intestinal proliferative cells. The latter forms the basis of an easy and rapid in vivo assay of somatic mutation (6) and also allows the fate of individual stem cells giving rise to such clones to be inferred. Hitherto these approaches have involved the use of different Dlb-1b and Dlb-1a homozygote strains, commonly C57BL/6J and SWR respectively, and this can create theoretical difficulties. For example, in chimaeras between C57BL/6J and SWR zygotes it is possible that the final distribution of cells in adult epithelium is influenced by metabolic or other differences between C57BL/6J and SWR derived cells. In addition, in the mutation assay large deletions and/or translocations which affect the activity of the Dlb-1b allele may also affect neighbouring heterozygous genetic loci. This may influence the subsequent fate of the resulting clone (eg if these genes were involved in intestinal differentiation). To ensure that these theoretical considerations will no longer apply, we have used conventional backcrossing procedures to breed a mouse congenic to C57BL/6J but possessing the genotype Dlb-1a/Dlb-1a which may be used in place of SWR. Materials and Methods Derivation of C57BL/6J Dlb-1a congenics. Conventional backcrossing procedures were employed. Briefly, C57BL/6J/Ola//Hsd males were mated with SWR/Ola//Hsd females to obtain C57BL/6J x SWR F1 Dlb-1b/Dlb-1a heterozygotes. The first backcross generation (F1) was created by crossing heterozygous males to C57BL/6J females. A small piece of tail was excised from males of F1 and subsequent litters and processed for histological sectioning and staining with DBA-Px. Positive staining of vascular endothelium in tail tip tissue distinguished Dlb-1b/ Dlb-1a heterozygotes (G+E+ phenotype) from Dlb-1b homozygotes (G+E-). The former were selected for backcrossing to C57BL/6J females for 12 generations. Six mice from generation F12, including at least one mouse from each litter, were sent to Harlan Olac Ltd and tested for isogenicity to C57BL/6J at 8 polymorphic loci by cellulose acetate electrophoresis (see Table 1). (The results shown for SWR are based on published alleles and were not determined experimentally). Generation of homozygotes. To generate homozygous Dlb-1a mice we set up five brother/sister matings of F12 siblings, the litters of which were designated F0. Rather than depend on random matings and retrospective identification of Dlb-1a homozygosity, we attempted to combine tai1 tip analysis (to identify and discard E- mice which are obligate Dlb-1b homozygotes) with the staining of faecal spreads to identify and discard G+ mice which includes both Dlb-1b homozygotes or Dlb-1b/Dlb-1a heterozygotes. Faecal spreads were obtained from F0 mice by introducing and withdrawing a small volume of sterile saline or in later experiments distilled water rectally via small bore tubing connected to a 1ml syringe. The contents of the syringe were air dried onto a microscope slide and subsequently fixed in 10% formol saline and stained with DBA-Px. Samples from SWR and C57BL/6J mice acted as negative and positive controls respectively. Samples were scored as being +/+, +/- or -/- for staining. C57BL/6J controls were invariably scored as +/+ or +/- and SWR as -/-. Therefore -/- F0 siblings were mated. After weaning F1 litters the F0 genotype was checked by DBA-Px on histological sections of gut (Figure 1). Results and Discussion The electrophoretic analysis of the six samples from F12 litters showed that all were isogenic to C57BL/6J in regard to the 8 polymorphic loci tested (Table 1). 12 F0 mice were chosen for breeding Dlb-1a homozygotes on the basis of the DBA-Px staining pattern in faecal spreads (G-) and tail tip sections (E+). As shown in Table 2 the G-E+ phenotype was subsequently confirmed in 7 by DBA-Px staining of gut sections including 3 breeding brother/sister pairs. The ratio 1:2:1 describes the distribution of Dlb-1 alleles in the F0 generation. However, Dlb-1 homozygotes were discarded on the basis of tail tip staining (E-). E+ mice included both heterozygotes and Dlb-1a homozygotes (ratio 2:l). Therefore the probability of randomly selecting a single Dlb-1a homozygote is 1 in 3 and of selecting 2 for breeding 1 in 9. Hence in using the prescreening procedure we were able to identify Dlb-1a homozygotes with a reasonable degree of accuracy (~70%). In fact the intensity of DBA-Px stained faecal spreads obtained from C57BL/6J mice was increased when the washing solution was changed from saline to distilled water. Consequently, if it were desirable to identify the activity of the Dlb-1b or Dlb-1a alleles in DBA-Px stained faecal spreads or tail tip sections respectively, for example in germline mutation studies, the resolution of the former could probably be improved. By crossing the newly established congenic Dlb-1a strains to conventional C57BL/6J mice Dlb-1b/Dlb-1a heterozygotes can be obtained. These mice will form the basis of an improved version of the Dlb-1 somatic mutation assay (7) in which mutations reflecting the single Dlb-1b allele are detected by observing DBA-Px negative staining clones in wholemount preparations of small intestine. Our suggested designation for the new strain is C57BL/6J-Dlb-1a. Acknowledgements. We thank Sue Ludgate, Linda Baldwin and Shirley Pease for their dedicated assistance. References 1. Uiterdijk HG, Ponder BAJ (1985). In 'Lectins' Vol IV (Eds TC Bog-Hansen, J Breborowicz). Walter de Gruyter & Co, Berlin, pp 161-168. 2. Uiterdijk HG et a1 (1986). Genet Res Camb, 41, 125-129. 3. Ponder BAJ, Wilkinson MM (1983). Dev Biol, 96, 535-541. 4. Ponder BAJ, Festing MF, Wilkinson MM (1985). J Embryo1 exp Morphol, 87, 229-239. 5. Ponder BAJ et a1 (1985). Nature, 313. 689-691. 6. Winton DJ, Blount MA, Ponder BAJ (1988). Nature 333; 463-466. 7. Winton DJ et a1 (1990). Cancer Res, 50, 7992-7996. TABLE 1: Isogenicity of backcrossed mice to C57BL/6J. Mouse No.: BC 57; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: BC 55; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: BC 51; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: BC 51; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: BC 51; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: BC 54; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: C57BL/6J/Ola//Hsd; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: a; Pgm-2: a; Es-1: a; Trf: b; Car-2: a; Apoa-1: b. Mouse No.: DBA/2/Ola//Hsd; Polymorphic loci: Hbb: d; Gpi-1: a; Pgm-1: b; Pgm-2: a; Es-1: b; Trf: b; Car-2: b; Apoa-1: b. Mouse No.: [SWR/Ola//Hsd; Polymorphic loci: Hbb: s; Gpi-1: b; Pgm-1: b; Pgm-2: a; Es-1: b; Trf: b; Car-2: b; Apoa-1: b]. TABLE 2 : Phenotypes(a) of F0 breeding pairs. Box No: 1; Male: G-E+; Female(a): G-E+; Box No: 2; Male: G-E+; Female(a): G-E+; Box No: 3; Male: G-E+; Female(a): G-E+; Box No: 4; Male: G+E+b; Female(a): G-E+; Box No: 5; Male: G+E+b; Female(a): G+E+b; Box No: 6; Male: NDc; Female(a): Ndc. a The phenotypes shown here are identified by DBA-Px staining of histological sections of gut and tail tips. G-E+ are Dlb-1a homozygotes, and G+E+ are Dlb-1b/Dlb-1a heterozygotes. b Mice incorrectly identified as G-E+ on the basis of DBA-Px staining of faecal spreads. c Not done. FIG 1. (Legend). Photomicrograph of small intestinal histological sections stained with DBA-Px and counterstained with Haemalum. (a) C57BL/6J/Ola//Hsd. Note positive staining in the villus (big arrow) and crypt (small arrow) epithelium and its absence in connective tissue. A small number of goblet cells which stain with greater intensity can be seen in the villus epithelium. (b) C57BL/6J-Dlb-1a . Note the absence of staining in epithelium. Positive staining is now present in blood vessels in the connective tissue core of villi and underlying crypts (arrows). |
