Regional characterization of a hamster–sheep somatic cell hybrid panel
Laboratoire de Pathologie Infectieuse et Immunologie, INRA-Tours, 37380 Nouzilly, France
Laboratoire de Ge´ne´tique Biochimique et de Cytoge´ne´tique, De´partement de Ge´ne´tique Animale, INRA, 78352 Jouy-en-Josas, France
Laboratoire de Biologie Mole´culaire et Ge´ne´tique, Universite´ Oran Es-Se´nia, 31000 Oran, Algeria
Received: 14 May 1999 / Accepted: 23 August 1999
Abstract. The regional characterization of a previously obtained
hamster–sheep hybrid panel is reported. Using data available from
ruminant maps (sheep, cattle, and goat), we have selected a set of
300 markers and have analyzed them by PCR in this hybrid panel.
Results obtained for 204 markers show the presence of all sheep
chromosomes (including gonosomes) in entire or fragmented form.
Analysis of syntenies has given 130 types of answer defining
segments of variable sizes. This study has led to the regional
characterization of this panel and provides comparative data on a
set of bovine and caprine markers. With the level of characteriza-
tion now achieved for this hybrid panel, the regional assignment of
new genes or markers to sheep chromosomes can be rapidly ob-
tained. Finally, this panel will help to collect new data for com-
parative mapping of domestic animals and to highlight the con-
servation of syntenic groups between closely related species, that
is, sheep, cattle, and goat.
Somatic cell hybrids have been an invaluable tool to improve maps
in various domestic species, such as cattle (Heuertz and Hors-
Cayla 1981), sheep (Saidi-Mehtar et al. 1981; Tucker et al. 1981),
or horses (Bailey et al. 1995). However, until recently, panels of
somatic cell hybrids were used only to obtain syntenic information.
While such data were the unavoidable starting point for building
new maps, present maps of the main domestic species (cattle and
pig) contain many more than 1000 markers (Rohrer et al. 1996;
Kappes et al. 1997; Barendse et al. 1997), indicating that somatic
hybrids will be useful only if they define subchromosomal regions.
Radiation hybrids constitute an improvement over “classical” so-
matic cell hybrids because they define small chromosome regions.
The donor cell line is exposed to highly energetic gamma irradia-
tion prior to fusion with a rodent cell line (Cox et al. 1990). By this
method, radiation hybrid panels have recently been constructed in
cattle (Womack et al. 1997). The major investment necessary to
build such a panel can, however, be partially avoided by exploiting
the spontaneous fragmentation occurring in the chromosomes of
the donor cell line. Indeed, the existence of such fragmented chro-
mosomes has been demonstrated in various cell hybrid panels.
Even without using a physical procedure to generate random chro-
mosome breaks, these spontaneous breaks are often sufficient to
considerably improve the resolution of a given panel. This possi-
bility has been used to characterize thoroughly a pig regional panel
(Yerle et al. 1996).
Previous analysis of genes (restriction fragment length poly-
morphism) and microsatellite markers (polymerase chain reaction,
PCR) has already enabled us to characterize a panel of 24 hamster–
sheep somatic cell hybrids at the chromosome level (Tabet-Aoul et
al. 1992, 1996). However, until now, the paucity of genome maps
for small ruminants has prevented the detailed description of the
chromosome composition of each cell line in this panel. This situ-
ation has now evolved with the recent development of genetic
maps for sheep and goats (de Gortari et al. 1998; Schibler et al.
1998), which have made available a wide panel of STR loci, some-
times associated with coding sequences. These markers are pre-
cisely mapped on the chromosomes, and their order is well de-
fined. This wealth of probes has now enabled us to characterize
regionally our cell hybrid panel, using 204 markers covering most
chromosomes. Among the selected markers, 22 were chosen near
genes, and 66 probes correspond to cosmids or BACs that have
been cytogenetically mapped by FISH to either cattle, sheep, or
goat chromosomes. This makes it possible to define physical
boundaries of several chromosomal segments, and to exploit com-
parative mapping data from the human genome.
We believe that with this characterization, our panel will be-
come an effective tool for localizing new probes rapidly on sheep
or goat chromosomes.
Materials and methods
Somatic cell DNA preparation.
Twenty-four hamster-sheep cell lines
(7 prepared with female ovine lymphocytes and 17 with male ovine fibro-
blasts) were previously obtained by Saı¨di-Mehtar et al. (1981). They were
cultured in minimal essential medium (MEM) supplemented with 1% an-
-glutamine, and 10% fetal calf serum. DNA was
extracted as described by Miller et al. (1988).
Choice of primers and PCR conditions.
Markers were selected from
the published sheep (Crawford et al. 1995; de Gortari et al. 1998), cattle
(Barendse et al. 1997), and goat (Schibler et al. 1998) linkage maps. They
comprised microsatellites, genes, and microsatellites associated with
genes. Primer sequences are available from the papers cited above and on
the web site http://sol.marc.usda.gov/genome.
PCRs were carried out on 75 ng of DNA in 25 l, with 0.5–1 U of Taq
DNA Polymerase (Gibco BRL, Life Technologies, UK), 1.5 m
dNTP in a Biometra UNO-thermoblock or a Perkin-Elmer Cetus
9600 thermocycler. After a 5-min denaturation at 95 °C, samples were
subjected to cycling for 30–35 cycles (30 s at 94 °C, 30 s at 55–63 °C, and
30 s at 72 °C). Annealing temperatures were sometimes adjusted for a
better sheep DNA amplification. PCR products were then loaded on 3%
agarose gels. Ovine and hamster genomic controls were systematically
included in the analysis of each microsatellite. To confirm our results, the
typing of discordant hybrids from the same syntenic group has been du-
Average lengths for each fragment were estimated by dividing the
number of markers in one fragment by the total number of markers ana-
lyzed and multiplying by the total length of ovine genome (3000 cM).
Syntenic groups were identified according to the rules proposed by Cheva-
let and Corpet (1986). With 24 hybrids, assignment to a syntenic group was
assumed for a correlation coefficient () superior or equal to 0.74.
Correspondence to: K. Tabet-Aoul
Mammalian Genome 11, 37–40 (2000).
© Springer-Verlag New York Inc. 2000