FLOW CYTOGENETICS OF DOMESTIC ANIMALS AND GENE MAPPING: WONDERS AND PITFALLS.

Bouvet A. Section of Medical Genetics, Veterinary Hospital, University of Pennsylvania, 3850 Spruce Street, Philadelphia. PA 19104-6010. USA

In the past five years, there has been great advances in the use of flow cytometry in gene mapping in domestic animals, but also some limitations in its use have started to emerge. Most of the progress has been noted in the pig flow karyotype although other livestock species have also been investigated.

Pig flow karyotype

Univariate

In 1986, univariate flow karyotypes for the pig were reported from France (2O) and Sweden (l3) and spot blot hybridization for a gene' located on chromosome 7 was described (l2). Since that time, Dr. Popescu and his group have been very active in standardizing the univariate pig flow Caryotype using various translocations (2,3).

Bivariate

Since pig chromosomes are few in number, have different length and different ratios of A-T and G-C rich areas, it has been possible to obtain a bivariate flow karyotype (9,10,14) following the method used for human chromosomes(ll). In the best preparations, twenty peaks would appear that would correspond to the 18 pairs of autosomes and the X and Y chromosomes.

The standardisation of the pig flow Caryotype was achieved as part of the European Pig Gene Mapping Project (PiGMaP)l. Indirect and direct methods used included comparison between male and female flow Caryotypes (6,24), comparison between relative chromosome length and relative fluorescence values for each peak(4,5,23), comparison of FACS profiles of pig-mouse hybrids cell lines containing pig chromosomes, dot blot hybridization(5), chromosome translocationS(7,24), PCR amplifications and chromosome painting using degenerated PCR to amplify DNA from flow-sorted chromosomes(17,18,21,29,30).

Problems

Blood cultures were an unreliable as source of constant good yield for seperation and now, a transformed cell line that has kept the normal chromosome complement is used(l6).

There is always a background of nuclear debris. Therefore, you can sort pieces of DNA with given fluorescence values that could be chromosome fragments, nuclear debris or clusters of small chromosomes(25).

The method used to amplify sorted chromosomes is more likely to amplify DNA repeats than single genes(l8,21).

To obtain a set of 20 filters for dot blot hybridization is very laborious, dot blots do not give fine localisation and background signal can be a problem.

Using FACS to characterize pig-rodent somatic cell hybrids and sort individuel pig chromosomes from these lines might be tricky. Chromosome rearrangements have been noted between mouse and pig chromosomeS(31), and loss of PGD gene in a seemingly intact pig chromosome 6 in a hybrid cell line have also been reported(3l). As reported in humans, the solution is to combine FACS sorting with chromosome painting(l9,28).

Future

Pig flow karyotyping is now geared toward sorting of chromosomes for making chromosome-specific libraries (22), and to generate chromosome-specific DNA repeats (8,15). These chromosome-specific probeswould be useful to screen genomic libraries, to characterize translocations and somatic cell hybrids panels and to study chromosome evolution and conserved synteny groups.

Other Species

Numerous identical chromosomes make it difficult for the equine, bovine and goat flow karyotypes, whereas in sheep the first three metacentrics could be sorted (9,10). However, another route can now be taken to generate chromosome-specific probes, chromosome microdissection. This has already been applied succesfully to bovine chromosomeS(27).

REFERENCES

1. Archibald A, Haley CS, Andersson L, Bosma AA, Davies W, Fredholm M, Geldermann H, Gellin J, Groenan M, Gustavsson 1, Ollivier L, Tucker EM & Van de Weghe A. 1991. PiGMaP: An European initiative to map the porcine genome. Anim Genet 22 Suppl 1:82-83.

2. Blaise F , Aycardi J, Boscher J & Popescu CP. 1990. Flow cytometry of normal and abnormal pig Caryotypes. Preliminary report. Ann Genet 33:146-151.

3. Blaise F, Aycardi J, Boscher J & Popescu CP. 1991. Flow cytometry in normal and abnormal pig Caryotypes. Genet Sel Evol 23 (supp 1):73s.

4. Bouvet A,, Konfortov B, Miller NGA, Brown D & Tucker EM. 1993. Identification of pig chromosomes in pig-mouse somatic cell hybrids flow Caryotypes. Cytometry 14:369-376.

5. Bouvet A, Konfortov B, Miller NGA, Miller RM & Tucker EM. 1993. Identification of pig chromosomes in bivariate flow Caryotypes by various methods. Anim Genet 24: (suppl 1), 84.

6. Bouvet A, Miller NGA &Tucker EM. 1992. Identification of chromosomes in pig flow Caryotype. Genet Res (Cambridge) 59:228.

7.Chardon P, Schmitz A, Chaput B, Frelat G & Vaiman M. 1991. SLA class 111 gene assignments combining chromosome sorting and polymerase chain reaction. Anim Genet 22 (suppl 1):86.

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10. Dixon SC, Miller NGA, Carter NP & Tucker EM. 1992. Bivariate flow cytometry of farm animal chromosomes: a potentiel tool for gene mapping. Anim Genet 23:203-210.

 11. van den Engh GJ, Trask BJ, Gray JW, Langlois RG & Yu LC. 1985. Preparation and bivariate analysis of suspensions of human chromosomes. Cytometry 6:92-100.

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14. Grunwald D. 1988. La cytogenetique en flux chez les grands mammiferes. Application chez le porc et le singe cynomulgus. In: La cytometrie en flux, pour l'etude de la cellule normale ou pathologique. Vol. 2. Techniques. (Metezeau PH, Ronot X, Lenoan-Merdrignac G & Ratinaud MH, eds). pp 277- 283. Mesi/McGraw-Hill, New York.

15.Hoyheim B, Keisrud A, Chaput B, Schmitz A, Frelat G & Davies W. 1993. Rapid isolation of porcine chromosomes specific microsatellites. Anim Genet 24:(In Press).

16. Kaeffer B, Bottreau E, Than LP, Olivier M & Salmon H. 1990. Histocompatible miniature pig model: selection of transformed cell lines of B and T lineages producing retrovirus. Int J Can 46:481-488.

17. Langford CF, Telenius H, Carter NP, Miller NGA & Tucker EM. 1992. Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes. Cytogenet Cell Genet 61:221-223.

18. Langford CF, Telenius H, Carter NP, Miller NGA & Tucker EM. 1993 Chromosome painting using chromosome-specific probes from flow-sorted porcine chromosomes. Anim Genet 24: (In Press).

19. Lengauer C, Riethman H & CremerT. 1990. Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and Ll primers, Hum Genet 86:1-6.

20. Matsson P, Anneren G & Gustavsson 1. 1986. Flow cytometric karyotyping of mammals, using blood lymphocytes: detection and analysis of chromosomal abnormalities. Hereditas 104:49-54.

21. Milan D, Yerle M, Schmitz A, Chaput B, Vaiman M, Frelat G &Gellin J. 1993. A PCR-based method to amplify DNA with random primers: determining the chromosomal content of porcine flow-karyotype peaks by chromosome painting. Cytogenet Cell Genet 62:139-141.

22 Miller JR, Dixon SC, Miller NGA, Tucker EM, Hindkjaer J & Thomsen PD. 2. A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica). Cytogenet Cell Genet 61:128-131.

23. Schmitz A, Chaput B, Fouchet P, Guilly MN, Frelat G & Vaiman M. 1992. Swine chromosomal DNA quantification by bivariate flow karyotyping and Caryotype interpretation. Cytometry 13:703-710.

24.Schmitz A, Chardon P, Gainche I, Chaput B, Guilly MN, Frelat G & Vaiman M.1992. Pig standard bivariate flow karyotype and peak assignments for chromosomes X, Y, 3 and 7. Genomics 14:357-362.

25. Telenius H, de Vos D, Blennow E, Willat LR, Ponder BA & Carter NP. 1993. Chromatid contamination can impair the purity of flow-sorted metaphase chromosomes. Cytometry 14:97-101.

26. Thomsen PD, Bosma AA, Kaufmann U & Harbitz 1. 1991. The porcine PGD gene is preferentially lost from chromosome 6 in pig X rodent somatic cell hybrids. Hereditas 115:63-67.

27. Thomsen PD, Telenius H, Miller JR & Christensen K. 1992. Bovine Y- chromosome-specific DNA probe generated by DOP-PCR using a sincle dissected Y chromosome as template. Anim Genet (In Press).

28. Trask BJ, van den Engh G, ChristensenM, Massa HF, Gray JW & Van Dilla M. 1992. Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescence in situ hybridization. Somatic Cell Mol Genet. 17:117-136.

29.Tucker EM, Langford CF, Bouvet A, Miller NGA, Telenius H & Carter NP 1992. Separation of pig chromosomes by flow cytometry and preparation of chromosome-specific probes. Proc 10th Europ Colloqu Cytogenet Domes Anim Utrecht, The Netherlands, August 22-28.

30. Yerle M, Milan D, Schmitz A, Vaiman M, Frelat G & Gellin J. 1993 . Definite characterization of porcine flow Caryotype by chromosome painting after PARM-PCR. Anim Genet 24: (In Press).

31. Zijlstra C, Bosma AA, de Haan NA, Thomsen PD & Tucker EM. 1992. Characterization of pig-rodent somatic cell hybrids. Proc. 10th Europ Colloqu Cytogenet Dom Anim. Utrecht, the Netherlands, August 22-28.


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