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Characterization of a Zebrafish/Mouse Somatic Cell Hybrid Panel

Mario Chevrette,* Lucille Joly,† Patricia Tellis,* Ela W. Knapik,‡ Jennifer Miles,†Mark Fishman,§ and Marc Ekker† , ¶ ,1

*Urology Division, Department of Surgery, McGill University and Montreal General Hospital Research Institute, Montreal, Quebec H3G1A4, Canada; †Loeb Health Research Institute, Ottawa Hospital, Ottawa, Ontario K1Y 4E9, Canada; ‡Institute of Mammalian

Genetics, GSF, Research Center for Environment and Health, Ingolstaedter Landstrasse 1, 85758 Neuherberg, Germany;§Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129; and

¶Department of Medicine and Department of Cellular and Molecular Medicine,University of Ottawa, Ottawa, Ontario K1H8M5, Canada

Received November 1, 1999; accepted December 29, 1999

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We have characterized a collection of zebrafish/mouse somatic cell hybrids with 211 genes and mark-ers chosen from the 25 zebrafish linkage groups.Most of the zebrafish genome is represented in thiscollection with 88% of genes/markers present in atleast one hybrid cell line. Although most hybridscontain chromosomal fragments, there are a few in-stances where a complete or nearly complete ze-brafish chromosome has been maintained in a mousebackground, based on multiple markers coveringthe entire chromosome. In addition to their use inmapping studies, this collection of somatic cell hy-brids should constitute an important tool as a sourceof specific chromosome fragments and for assessingthe function of genome regions. © 2000 Academic Press

The zebrafish Danio rerio has become one of themost popular organisms with which to study embry-onic development of vertebrates. This is largely dueto the characteristics of this organism that facilitatedevelopmental and genetic analyses (17, 18). Ze-brafish are prolific (a few hundred eggs per spawn-ing), and their generation time is short (2–3 months).The embryos are optically clear and easily accessible,and their development is rapid, all properties thatfacilitate the detection of mutations. Two large-scalescreens for mutations that affect development haveresulted in the identification of about 2000 muta-tions (6, 9). The defects encompass all aspects oforgan development and embryonic patterning andalso include behavioral mutants. Several mutantphenotypes closely resemble human diseases. Forexample, the sauternes and yquem mutations in ze-

rafish were assigned to genes whose human homo-

1 To whom correspondence and reprint requests should be ad-dressed. Telephone: (613) 798-5555, Ext. 6033. Fax: (613) 761-5036.

bE-mail: mekker@lri.ca.

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ogues are deficient in X-linked congenital sider-blastic anemia and hepatoerythropoietic porphyria,espectively (3, 28). The collection of mutants consti-utes a most valuable resource, and the zebrafish willlearly become one important model with which totudy human diseases. However, most of the (mu-ated) genes resulting in these developmental phe-otypes are yet to be identified. This important stepill be greatly facilitated by dense maps of the ze-rafish genome and other genomic resources.Genetic maps of the zebrafish have been produced

sing random amplified polymorphic DNA (RAPD)arkers, simple polymorphic sequence repeats

SSRs), cloned genes, and expressed sequences (10,4, 19, 22, 23, 25). Determining the position of newenes or anonymous markers on these maps requireshe presence of polymorphisms between zebrafishtrains. Somatic cell hybrids and radiation hybridsrovide fast and reliable methods for assigning geneso their chromosomal locations without the need forolymorphism. Two radiation hybrid maps of theebrafish genome have been recently reported (11,3) and are intensely used to position cloned genesnd ESTs.We have previously generated a collection of somatic

ell hybrids by fusing zebrafish fibroblasts with aouse melanoma cell line (7, 8). This somatic hybrid

anel was used to anchor one of the two meiotic mapsf the zebrafish genome (19). This somatic cell hybridanel will be useful as a source of DNA for specifichromosomal regions, to bridge chromosomal regionsot adequately resolved in one of the two radiationybrid panels, and to assess the function of specifichromosomal regions. Here we report the properties of2 hybrids of this somatic panel, characterized with11 markers located on the 25 different linkage groups.The production of zebrafish/mouse somatic cell hy-

rids between zebrafish ZF4 or LFF embryonic fibro-

Genomics 64, 119–126 (2000)doi:10.1006/geno.1999.6124

0888-7543/00 $35.00Copyright © 2000 by Academic Press

All rights of reproduction in any form reserved.

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blasts and mouse melanoma cells has been described(7, 8). The zebrafish/mouse hybrids called ZFB andLFFB are derived from the ZF4 and LFF zebrafishparental cells, respectively. Hybrid cells were main-tained in DMEM/F12 1 10% fetal bovine serum 1 400mg/ml of G418. Both cells and DNA samples from thissomatic cell hybrid collection are available from theauthors upon request.

Two hundred eleven markers including clonedgenes (23), SSRs (available from Research Genetics)(12, 19), and cloned zebrafish RAPDs (22) have beenused to map each hybrid of this panel. PCR amplifi-cation was performed as described (8).

FIG. 1. Characterization of the zebrafish/mouse somatic cell hybon the top of the thick vertical lines. The markers analyzed are indicamarkers on the genetic map is indicated on the right. SSR and Z marare indicated by the vertical lines with the hybrid number indicatedexact position of breakpoints in each chromosomal fragment is notpositive marker and the first negative marker.

Ninety-two cell lines, 79 ZFB and 13 LFFB, were

chosen for further analysis, after a preliminary char-acterization by PCR. This choice was made to elim-inate lines that appeared to have identical chromo-some content and may correspond to colonies thatoriginate from a single clone that had duplicatedshortly after fusion.

The results of the characterization of the collectionof zebrafish/mouse somatic cell hybrids with 211markers is shown schematically in Fig. 1. A table ofmarker retention by cell line can be seen at thefollowing Web site: http://zfish.uoregon.edu/ZFIN/.The positions of the markers on the 25 linkagegroups are based on the zebrafish meiotic maps (19,

collection with 211 markers. Linkage groups (1 to 25) are identifiedon the left of each linkage group, while the distance (in cM) between

s are microsatellite markers. DNA fragments present in each hybridove. A space indicates the absence of a particular marker. Since theown precisely, the vertical lines extend midway between the last

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22). We assayed roughly equivalent numbers of

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markers for each linkage group and tried to selectmarkers so that the corresponding chromosomeswere evenly covered, as inferred from genetic dis-tances.

The percentage of the 211 markers that we coulddetect in at least one hybrid was, on average, 88%,giving us an estimate of the percentage of the ze-brafish genome that could be successfully trans-ferred during a whole-cell fusion with a mouse cellline. Therefore, there are still regions of zebrafishchromosomes that were not retained in our hybrids,despite the fact that a total of three fusion experi-ments were performed, with two different zebrafishcell lines. This includes, for example, the 2.4-cMinterval encompassing the wt2 gene and the z1145microsatellite marker on LG 10. It is unlikely thatsuch chromosome regions failed to be transferredbecause of a deleterious effect that one or severalgenes from this region might have on growth or

FIG. 1—

survival of B78 cells. Indeed, markers from this

region were successfully transferred to the same re-cipient cells in the LN54 radiation hybrid panel (13).We cannot exclude the possibility that the combina-tion of genes present on the same chromosome wouldimpede the growth of B78 cells. This would explainwhy some regions of zebrafish chromosomes are notpresent in this whole-cell hybrid panel but are rep-resented in radiation hybrids, where chromosomesare more fragmented. Finally it must be mentionedthat neither the present somatic cell hybrid panelnor the two radiation hybrid panels for the zebrafishshow 100% coverage of the genome. Therefore, it isstill possible that zebrafish genes that might func-tion as tumor suppressor genes (B78 is a melanoma),cell growth regulator genes, or senescence-inducinggenes and that might be transcribed in the resultinghybrid would prevent their growth.

The overall retention of the hybrid panel was 4.5%;that is, for each hybrid, 4.5% of the zebrafish ge-

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nome, as represented by the 211 chosen markers,

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was retained. This value was not uniform for each ofthe 25 linkage groups and varied between 1.6% forLG 19 and 10.9% for LG 20 (Fig. 2). It is worthmentioning that both in this hybrid panel and in theLN54 radiation hybrid panel (13), LG 20 was partic-ularly well retained. Thus, in contrast with regionsthat could not be transferred, perhaps due to detri-mental effects on the growth or survival of the recip-ient B78 cells, it is possible that transfer of frag-ments of the chromosome corresponding to LG 20may have some beneficial effects. It is unlikely thatpreferential retention of LG 20 could be attributableto the presence of the drug selection gene used to tagzebrafish chromosomes in the donor cells becausethree different zebrafish cell lines were used to makethe two panels. Moreover, the selectable marker wasrandomly introduced by transfection in all three cellpopulations, and, in each case, more than 400 differ-ent clones were used as a pool for the fusion with B78

FIG. 1—

cells.

Some hybrids seem to contain a complete zebrafishchromosome. For example, all nine markers testedon LG 7 were present in two hybrids (see LFFB1 andLFFB3 in Fig. 1). Although we realize that theseseven markers may not cover the whole chromosome,and thus not exclude some small deletions, it shouldbe noted that they were located on both arms of thischromosome (19, 23) and separated by a total of 90cM on the genetic map. Similarly, all seven markersof LG 3 were present in the ZFB212 hybrid, whileboth LFFB3 and LFFB10 contained all eight mark-ers (45 cM apart) of LG 18. Since fluorescence in situhybridization (FISH) analysis using whole zebrafishpainting probe rarely detected whole zebrafish chro-mosomes standing by themselves in hybrids (7, 8), itis possible that complete or nearly complete ze-brafish chromosomes could be integrated betweenmouse chromosomal elements.

During the preliminary characterization of this

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hybrid panel, we had used total zebrafish genomic

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DNA probe in FISH on hybrid chromosome spreadsto estimate the number of zebrafish chromosomespresent in each hybrid. These analyses seemed toindicate that hybrids contained between 0 and 4zebrafish chromosomes or chromosome fragmentsdetectable by FISH (4). This contrasts with the num-ber of human chromosomes retained in human/mouse hybrids generated with the same B78 recipi-ent. Indeed, in a similar fusion between human skinfibroblasts and B78 melanoma cells, hybrids contain-ing as many as 19 human chromosomes were foundupon FISH analysis (26). PCR analysis of the ze-brafish/mouse hybrids shows that hybrids have in-corporated more zebrafish elements than previouslydetermined by the FISH analysis. In fact, some hy-brids had zebrafish fragments coming from as manyas 11 different linkage groups (4, 8). Therefore, ei-

FIG. 1—

ther some of the elements are small enough to escape

detection by FISH or, more likely, segments from twodifferent chromosomes have integrated together in-side a mouse chromosome.

The panel presented here was used to identifybmp2 and tolloid as the genes affected by the swirland mini fin mutations, respectively (5, 21). Thepanel was also used to assign hox genes to theirrespective clusters and was instrumental in theidentification of additional hox clusters in the ze-brafish (1, 24). Recently, two radiation hybrid panelshave become available for the zebrafish (11, 13), andthey offer mapping with considerably higher resolu-tion than the somatic cell hybrids described here.Nevertheless, some of the hybrids described in thepresent study contain what seems to be intact chro-mosomes and could be useful in bridging regions ofthe zebrafish genome not resolved in one of the ra-

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diation hybrid panels.

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The greatest advantages of the somatic cell hy-brids described here reside in their use as a source ofDNA for specific zebrafish chromosome regions andfor assessing the function of genomic regions. Thezebrafish chromosomes in the donor ZF4 and LFFcells used for hybrid production were “tagged” withthe neomycin resistance gene introduced at random.Pools of more than 400 stable transfectants wereused in each fusion. Therefore, it is likely that allzebrafish chromosomes have been tagged with thedrug resistance gene. We previously showed thatonce cells are cultured in the presence of the drugselection agent, the tagged zebrafish chromosome (orchromosome fragment) is selectively maintained atthe expense of the other, presumably untagged, ze-brafish chromosomes. It is thus possible to culturesome of the hybrids and specifically select for onespecific chromosome. The resulting subclone can be

FIG. 1—

used as a source of DNA for this zebrafish chromo-

some, for example, by inter-DANA/mermaid PCR (8).Furthermore, with a selection scheme similar to thatused to produce the hybrids, one is able to transferthe tagged chromosome (or chromosomal fragment)into another recipient cell and thus assess the func-tion of the genes transcribed from this region. Ininterspecies hybrids, the transferred chromosomesusually conserve the transcription properties theyhad in the original cells, in this case zebrafish fibro-blasts. This approach has already been used, in otherspecies, to clone an enzymatic regulator (15) andpinpoint the location of tumor suppressor (2, 20) andsenescence-inducing genes (16, 27).

ACKNOWLEDGMENTS

This work is supported by a grant from the Medical ResearchCouncil of Canada to M.E. and M.C. and by NIH Grant DK-558383to M.F. We thank Howard Jacob and William Talbot for providing

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results prior to publication.

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