Separation of pig chromosomes by flow cytometry and preparation of chromosome-specific probes

E.M. Tucker', C.F. Langford', A. Bouvet', N.G.A. Miller, H Telenius, N.P. Carter2 and P.D. Thomsen3  

'AFRC Institute of Animal Physiology and Genetics Research, Babraharn, Cambridge (U.K.), 2Department of Pathology, University of Cambridge, Cambridge (U.K.), 3Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, Copenhagen (Denmark).

Independent studies by us (Dixon et al., 1992) and colleagues in France (Grunwald et al., 1986) have demonstrated that the bivariate flow karyotype of the male pig shows 20 clusters, each cluster presumably representing one of the autosome pairs plus X and Y. More recently, attention has been directed towards the identification of each of the clusters. The two largest chromosomes, Nos. 1 and 13, and the smallest, No. 18 are easily recognized by size alone; X and Y are identified by comparing the flow karyotypes of male and female pigs (Bouvet et al., 1992). These five clusters were positively identified by chromosome painting using the DOP-PCR (degenerate oligonucleotide-primed polymerase chain reaction) technique (Telenius et al., 1992a & b, Langford et al., 1992) to amplify and biotinylate DNA from sorted chromosomes. In this report we describe the preparation by this technique of probes which when painted on to normal mŽtaphase spreads gave positive identification of each of the chromosome clusters.

Methods. Pig chromosomes prepared from cultured peripheral blood lymphocytes were labelled with Hoechst 33258 and chromomycin A3 and sorted on a dual laser FACStar Plus flow cytometer (Becton Dickinson) as previously described (Dixon et al., 1992).

300-500 chromosomes from each cluster of the flow Caryotype were isolated by sorting directly into PCR tubes containing distilled water.

DOP-PCR was then carried out as described by Telenius et al. (1992a,b) and Langford et al. (1992), using the primer 6-MW(5'-CCGACTCGAGNNNNNNATGTGG-3') and Taq-polymerase. The PCR products were labelled by re-amplifying at a high annealing tempŽrature, incorporating biotin- 1 1 -DUTP (Sigma).

In situ hybridization and detection were performed according to Pinkel et al. (1986, 1988) on pig metaphase spreads prepared from peripheral blood lymphocytes or on metaphase spreads of chromosomes from pig/mouse somatic cell hybrids. Briefly, the cells were cultured in the presence of phytohaemagglutinin, blocked with methotrexate and then treated with 5-bromodeoxyuridine followed by colcemid. Metaphase spreads were prepared and in situ hybridization carried out using the biotinylated probes. The hybridized probes were detected by treating the slides with avidin-FITC, then with biotinylated anti-avidin followed by avidin FITC again. They were counterstained with DAPI(4'-6'-diamidino-2-phenylindole) and PI(propidium iodide), (Verma and Babu, 1989). The slides were analysed using a confocal laser scanning microscope (MRC-600; BioRad Microscience Ltd).

Results . Biotinylated probes were prepared from chromosomes sorted from each of the 20 clusters seen in the flow karyotype of a male pig (Fig. la).

The probes were painted onto pig metaphase spreads and the labelled chromosome identified by its R-banding pattern visualised by the DAPI-PI stain (Fig. lb), Figure lc shows chromosome No. 10 highlighted by the use of the chromosome "paint" prepared from the sorted chromosomes.

The "paints" were also used to detect pig chromosomes in pig/mouse somatic cell hybrids. The pig flow Caryotype with all the clusters identified is shown in Fig. la.

Discussion . By means of both indirect and direct methods it has been possible to identify each of the clusters seen in the bivariate flow karyotype of the pig. Our results agree well with those of independent parallel studies carried out in France (Yerle et al., 1992) and therefore we can now establish with reasonable certainty a standard bivariate flow karyotype for the pig and thus open the way for the isolation of individuel pig chromosomes for the PiGMap project. The chromosome-specific paints should also be useful for the study of translocations and for comparative mapping as well as for identification of pig chromosomes in somatic cell hybrids.

References

Bouvet, A., Konfortov, V., Miller, N.G.A., Miller, J.R. & Tucker, E.M. (1992) identification of porcine chromosomes in bivariate flow Caryotypes using various methods. Proc. XIIII Internat. Conf. Animal Genetics, Interlaken, Switzerland. Anim. Genet.24 (suppl 1):84 .

Dixon, S.C., Miller, N.G.A., Carter, N.P. & Tucker, E.M. (1992) Bivariate flow cytometry of farm animal chromosomes - a potentiel tool for gene mapping. Anim. Genet. 23, 203-210.

Grunwald, D., Geffrotin, C., Chardon, P., Frelat, G. & Vaiman, M. (1986) Swine chromosomes: flow sorting and spot blot hybridisation. Cytometry 7, 582-588.

Langford, C.F., Telenius, H., Carter, N.P., Miller, N.G.A. & Tucker, E.M. (1992) Chromosome painting using chromosome-specific probes from flow sorted pig chromosomes. Cytogenet. Cell Genet. (in press).

Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J. & Gray, J. (1988) Fluorescence in situ hybridisation with human chromosome-specific libraries: dŽtection of trisomy 21 and translocations of chromosome 4. Proc. natl. Acad. Sci. U.S.A. 85, 9138-9142.

Pinkel, D., Straume, T. & Gray, J.W. (1986) Cytogenetic analysis using quantitative, high sensitivity fluorescence hybridisation. Proc. natl. Acad. Sci. U.S.A. 83, 2934-2938. Telenius, H., Carter, N.P., Bebb, C.E.

Nordenskj™ld, R., Ponder, B.A.J. & Tunnacliffe, A. (1992a) Degenerate ligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13, 718-725.

Telenius, H., Pelmear, A.H., Tunnacliffe A., Carter, N.P., Behmel, A., Ferguson-Smith, M.A., Nordenskj™ld, M., Pfnagner, R. & Ponder, B.A.J. (1992b) Cytogenetic analysis  by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chrom. Cancer 4, 257-263.

Verma, R.S. & Babu, A. (1989) Human Chromosomes: Manual of Basic Techniques, pp 115-116 (Pergaman Press, Oxford/New York).

Yerle, M., Milan, D., Schmitz, A., Vaiman, M., Frelat, G. & Gellin, J. 0 (1992) Definite characterization of porcine flow Caryotype by chromosome painting after PARM- PCR. Proc. XHI Internat. Conf. Animal Genetics, Interlaken, Switzerland. Anim. Genet. (in press).

This work was supported by grants from the AFRC, EC (PiGMaP project) and the Cancer Research Campaign. H.T. is supported by the Swedish Medical Research Council, The Torsten and Ragnar Soderberg Foundations, The Crafoord Foundation and the Gyllenstiernska-Krapperup Foundation.

Legend to Figure 1

a)Bivariate flow karyotype of male pig with all the chromosome clusters identified.

b)Normal pig metaphase spread. Left side R-banding pattern visualised by DAPI/PI stain. Right side, the same spread showing FITC-labelled chromosome No. 15 (indicated by arrows).

c)Normal pig metaphase spread showing chromosome No. 10 labelled with fluorescent probe


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