Proc. Nat. Acad. Sci. USAVol. 69, No. 10, pp. 2757-2761, October 1972

Studies of Mice with a Balanced Complement of 36 Chromosomes Derivedfrom F1 Hybrids of Tlih and TIAld Translocation Homozygotes

(Robertsonian translocations/mouse hybrid nondisjunction/quinacrine mustard banding/Giemsa banding)

BEVERLY J. WHITE, JOE-HIN TJIO, LISA C. VAN DE WATER, AND CLARE CRANDALL

Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes ofHealth, Bethesda, Maryland 20014

Communicated by Theodore T. Puck, June 29, 1972

ABSTRACT F, hybrids with 38 chromosomes, in-cluding single T1Wh and TIAld translocations, resultedwhen mice homozygous for the Robertsonian transloca-tions T1Wh and TiAld were crossed. Meiotic studies of thehybrids showed two trivalents, indicating nonhomologyof the T1Wh and TIAld chromosomes. The hybrids hadfrequent (25%) unbalanced meiotic metaphase II comple-ments; one trisomic mouse resulted from six F1 crosses.The F, crosses also produced one mouse with 36 chromo-somes homozygous for both T1Wh and TIAld, as well asmice with balanced polymorphic complements of 37, 38,39, and 40 chromosomes. By crossing the F2, a homoge-neous line with 36 chromosomes was established. The lineis phenotypically normal, fertile, and has balanced meio-tic metaphase II complements. Analysis of the chromo-somes of these mice with quinacrine mustard and theGiemsa-banding technique confirmed that T1Wh andTIAld consisted ofchromosomes 5;19 and 6;15, respectively.

Crosses between this line and other existing transloca-tion stocks may produce new strains of mice with evenfurther reduction in chromosome number. Accumulationof Robertsonian translocations, a possible evolutionarymechanism in the wild, can be studied in the laboratory.F1 hybrids from certain crosses are also an importantmodel for human translocation carriers; both have similarmeiotic abnormalities and often have aneuploid offspring.

Four different Robertsonian translocations have now beendescribed in mice (1-4). Mice homozygous for these transloca-tions carry two biarmed chromosomes and have a totalchromosome number of 38, while normal mice have 40 acro-centric chromosomes. The autosomes involved in most of thesetranslocations have been identified by analysis of specificbanding patterns after staining with quinacrine mustard.However, some of the chromosome numbers have been re-assigned after correlation of banding patterns with lengthmeasurements (5). The TlWh translocation chromosome(TW) is formed by fusion of autosomes 5 and 19 (6), while theT163H translocation involves chromosomes 9 and 19 (5, 7, 8).The TIAld translocation chromosome (TA) consists of auto-somes 6 and 15 (8). The two arms of the THEM translocationhave been tentatively identified as chromosomes 8 or 9 and16 or 17 by length measurements (4).Reported here are studies of progeny from crosses between

TlWh and TlAld homozygotes. The purposes of the studywere to confirm the identity of the TW and TA translocationchromosomes, to study meiotic disjunction of the F1 (TW/

+ TA/+) hybrids, and to study the F2 offspring for balancedand unbalanced complements. One trisomic mouse was found,and one homozygous (TW/TW TA/TA) mouse was produced.By selecting progeny from certain crosses, a phenotypicallynormal and fertile line of mice with 36 chromosomes wasestablished.

METHODS

Animals. Mice homozygous for TlAld (TA/TA) were ob-tained from Dr. A. L6onard, Centre d'Etude de l'EnergieNucl6aire, Mol, Belgium. TlWh homozygotes (TW/TW)were from stock maintained by our laboratory. Eight F1litters from crosses between (TW/TW) females and (TA/TA)males were used for meiotic studies and F1 crosses. 41 F2progeny were karyotyped, and certain F2 mice were crossedto derive additional (TW/TW TA/TA) mice (Table 1). 22Progeny from these F2 crosses were then karyotyped. Forcrosses of F1 males and females with nontranslocation-bearinganimals, AKR/J mice from the Jackson Laboratories, BarHarbor, Me. were used.

Chromosome Preparations. Meiotic metaphase I(M I) andII (M II) cells from testicular preparations (3) from 29 F1males were analyzed. All progeny were karyotyped from 72-hrsuspension cultures of spleen removed without killing theanimal (3). Direct preparations were used to karyotype fetusesof 11-17 days gestation from the crosses of F1 with AKR/Jmice. Similar preparations of 15-day fetuses were used todetermine the banding patterns of the TW and TA chromo-somes with quinacrine mustard and the Giemsa-banding dif-ferential staining techniques. The method of Caspersson et al.(9) was used for quinacrine mustard studies. For Giemsa-banding, the acetic-saline-Giemsa technique of Bucklandet al. (10) was used. Karyotypes were arranged according tothe Committee on Standardized Genetic Nomenclature forMice (5).

RESULTSProgeny of crosses between (TW/TW) females and(TA/TA) males

51 Mice resulted from eight matings. The average litter size atbirth was 6.4 (Table 1), and litter size ranged from four tonine. Karyotypes from F1 spleen cultures showed 38 chromo-somes, including the submetacentric TW and TA transloca-tions (Fig. la). Analysis of 722M I cells from 29 F1 males con-sistently showed 15 autosomal bivalents, an XY bivalent, and

Abbreviations: TA and TW, TlAld, and TlWh translocationchromosomes, respectively; M I, meiotic metaphase I; M II,meiotic metaphase II.

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TABLE 1. Progeny from crosses between T1Wh and TiAld

No. of miceAverage karyotyped

Total litter Chro-No. of no. of size at Fe- mosome Translocationlitters mice birth Male male no. chromosomes

38(TW/TW) 9 X 38(TA/TA) '8 Fi 51 6.4 20 25 38 TW/+ TA/+

38(TW/+ TA/+) 9 X 38(TW/+ TA/+) CP6 F2 43 7.2

0 1 36 TW/TW TA/TA5 2 37 TW/+ TA/TA0 1 37 TW/TW TA/+4 5 38 TW/+ TA/+1 0 38 TW/TW +/+2 3 38 +/+ TA/TA6 1 39 TW/+ +/+3 4 39 +/+ TA/+0 2 40 +/+ +/+0 1 39 TW/+ TA/+*

37(TW+ TA/TA) 9 X 37(TW/+ TA/TA)e2 16 8.0

1 0 36 TW/TW TA/TA2 1 37 TW/+ TA/TA0 1 38 +/+ TA/TA

36(TW/TW TA/TA) X 37(TW/+ TA/TA) e1 9

0 4 36 TW/TW TA/TA1 1 37 TW/+ TA/TA

38(TW/TW +/+) 9 X 38(TW/+ TA/+)e2 16 8.0

2 37 TW/TW TA/+1 38 TW/TW +/+1 38 TW/+ TA/+1 39 TW/+ +/+

37(TW/TW TA/+).9 X 37(TW/+ TA/TA)e1 8

1 36 TW/TW TA/TA2 37 TW/TW TA/+3 38 TW/+ TA/+

TA and TW = T1 Ald and TlWh translocation chromosomes,respectively.

*Trisomy'19.

two trivalents (Fig. 2a). One trivalent consisted of the TWchromosome associated with the autosomes homologous toits short and long arms, and the other included the TA chro-mosome paired with its homologues.

If the two trivalents undergo balanced disjunction inde-pendently of each other, the following types of M II comple-ments would occur in equal numbers: (1) cells with a chromo-

TABLE 2. Metaphase II complemnts of29(TW/+ TA/+) males

No. oftrans- No. of

Chromo- location chromo- Cells countedsome chromo- someno. somes arms Number Percent

17 0 17 2 0.417 1 18 6 1.217 2 19 4 0.818 1 19 29 5.618 2 20 71 13.8*19 0 19 28 5.419 1 20 220 42.6*19 2 21 12 2.320 0 20 95 18.4*20 1 21 41 7.920 2 22 5 1.021 1 22 3 0.6

Total 516 100.0

* Balanced complements total 74.8%.

CC

FIG.1. (a) Spleen metaphase of an F1 38(TW/+ TA/+) maleshowing the TA and the TW submetacentrics (arrows). (b) Meta-phase from a newborn female with 39 chromosomes, 41 chromo-some arms, and trisomy (arrows) for number 19.

some number of 18 including TW and TA, (2) cells with acount of 19 including TW, (3) those with a count of 19 includ-ing TA [in practice, types (2) and (3) usually could not bedistinguished], and (4) cells with 20 acrocentrics. The actualcounts deviated from this (Table 2), and while the expectedbalanced complements predominated (74.8%, see also Fig.3a, b, and c), many of the cells (25.2%) appeared to be theresults of nondisjunction (Fig. 3d). It is therefore theoreticallypossible for the F1 to produce zygotes with duplication ordeficiency for one or more of the chromosomes involved in thetranslocations (chromosomes 5, 6,15, or 19).

Results of other crosses

The average size of the six F2 litters was 7.2 (range 3-10).41 of 43 F2 survived and were karyotyped (Table 1). Accordingto the M II analysis, balanced progeny with a polymorphicseries of 36, 37, 38, 39, or 40 chromosomes should be found.The karyotype 38(TW/+ TA/+) should be most frequentand karyotypes 36(TW/TW TA/TA), 40(+/+ +/+), 38-(TW/TW +/+), and 38(+/+ TA/TA) least frequent. Theactual results (Table 1) were consistent with this, althoughthere were fewer animals of karyotype 37(TW/TW TA/+)than expected. One nonviable female F2 mouse had 39 chromo-somes, includingTW and TA (Fig. lb). The number of chro-mosome arms was 41, and trisomy for chromosome 19 was

present. In this instance, trisomy was apparently the result ofnondisjunction of theTW trivalent chromosomes.The results of F2 crosses are shown in Table 1. Most of the

stock with 36 chromosomes resulted from a cross between a

(TW/TW TA/TA) female and a 37(TW/+ TA/TA) male.Animals of karyotype 36(TW/TW TA/TA) were phenotypi-

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FIG. 2. (a) Meiotic metaphase I from an F1 male with 15autosomal bivalents, an XY bivalent, and two trivalents (arrows).(b) A 36(TW/TW TA/TA) male diakinesis with 18 bivalents.Note two large rings (arrow8) formed by the paired TW and TAchromosomes.

cally normal and fertile; the average size at birth of ten (TW/TW TA/TA) litters was 6.1 (range 2-10).

Meiosis of males with 36 chromosomes

Cells (1082 M I and 310 M II) from 13 (TW/TW TA/TA)males were analyzed. At M 1, 17 autosom'al bivalents plus anXY bivalent were consistently present. Two large rings werefrequent (Fig. 2b), typical of the single large ring of maleshomozygous for a single Robertsonian translocation (1, 3).Balanced M II cells from (TW/TW TA/TA) males shouldhave a count of 18, including both TW and TA. Of the 310M II cells studied, 301 were 'apparently balanced (97.1%) and9 (2.9%) appeared to be damaged during processing, sincetheir counts were less than 18. Cells with counts greater than18, indicating nondisjunction, were not present.

CrossesbetweenF,and AKR/JThese crosses are described in Table 3. In the six crosses

between (TW/+ TA/+) females and AKR/J males, 83.8%of corpora lutea were represented by implants and 82.3%of the total implants appeared to be viable fetuses. A lowerproportion of corpora lutea resulted in 'implants (60.0%) andfewer of the total implants appeared viable (69.0%) when theAKR/J females and F1 males were crossed. All fetuses appear-ing viable were successfully karyotyped, and no unbalancedcomplements were observed. Our M II studies of (TW/+-

balanced karyotypes could be expected in a cross with normalmice. The results shown in Table 3 were consistent with this,although there was greater deviation from the expected1: 1:1:1 ratio in the six crosses of the F, females with normalmales than in the crosses of AKR/J females with F1 males.

Differential staining studies

The quinacrine mustard karyotype of a male 36(TW/TWTA/TA) fetus is shown in Fig. 4. The TW chromosomes showbanding patterns typical for chromosome 5 (long arm) and 19(short arm) (5). The banding patterns of the arms of TA areconsistent with those described by Miller et al. (8) and by theCommittee on Standardized Genetic Nomenclature for Mice(5), for chromosomes 6 (long arm) and 15 (short arm).Giemsa-banded karyotypes of (TW/TW TA/TA) mice

(Fig. 5) indicated that in general, the Giemsa- and quinacrinemustard-bands coincide, and confirmed the identity of TWand TA as chromosomes 5; 19 and 6; 15, respectively. Inmetaphases with prominent heterochromatic staining near thecentromeres (C-bands), these regions of both TW and TAappeared double, a finding consistent with the theory thatRobertsonian translocations result from centric fusion, with-out loss of paracentromeric heterochromatin.

DISCUSSIONMeiotic studies of F1 males from crosses between (TW/TW)females and (TA/TA) males confirmed that TW and TA donot share a chromosome in common. Two trivalents at M Iindicated nonhomology of the arms of TW and TA, in con-trast to previous meiotic studies of F1 males from crosses be-tween TlWh and T163H homozygotes (6), where the chainquadrivalent atM I showed that TlWh shared a chromosomein common with T163H (number 19). In both cases, meioticstudies were consistent with identification of the chromosomesby differential staining methods (6-8).The significant proportion of unbalanced M II complements

in (TW/+ TA/+) F1 males contrasts with the low percentageof such cells in males heterozygous for TlWh or T163H alone

TABLE 3. Crosses of (TW/+ TA/+) F1 with AKR/J miceat 11-17 days gestation

F19 F. crX X

Cross AKR/J a" AKR/J 9

Number of crosses 6 6Total corpora lutea 74 70Total resorptions 11 12Necrotic fetuses 0 1Total viable implants 51 29Total implants 62 42% of total implantsviable 82.3 69.0

% of corpora lutearepresented by im-plants 83.8 60.0

Karyotypes38(TW/+ TA/+) 9 (3d 69) 7 (4e 39)39(TW/+ +/+) 15 (6d' 9 Q) 7 (4d'3 9)39 (+/+ TA/+) 8 (4"49 ) 7 (2 5 Q9)40(+/+ +/+) 19 (10d 9Q9) 8 (4'49)

Total karyotyped 51 (236' 28 9 ) 29 (146' 15 9 )TA/+) males indicated that equal numbers of a series of four

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a

C d~~{ s o - e- .rcf

C ~~~~~~~dFIG. 3. Meiotic metaphase II cells from F1 with chromosome numbers of: (a) 18 including TW and TA, (b) 19 including one trans-

location, (c) 20, and (d) 20 including one translocation.

I a v~~~~~I

FIG. 4. Quinacrine mustard karyotype of a 36(TW/TW TA! FIG. 5. Giemsa-banded karyotype of a 36(TW/TW TA/TA)rA) male identifying the TW chromosome as 5;19 and the TA male indicating similarity of the quinacrine and Giemsa-bandingI]a 6;15. patterns.

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(1, 3). These singly heterozygous males had a single trivalentat M I and no detectable trisomic progeny. Similar studies ofTlAld heterozygotes have not been reported. Baranov andDyban (11) found that male T11EM heterozygotes had onlybalanced progeny in crosses with normal female mice termi-nated at 8 days of gestation. However, heterozygous femalescrossed with normal males produced 14.3% trisomic embryos.The tendency for only female T11EM to have unbalanced off-spring is similar to that of humans carrying Robertsoniantranslocations. Female D;G, translocation carriers have asignificantly greater frequency of trisomic offspring than malecarriers, while data on families in which G;G and D;D trans-locations are segregating are not extensive enough to comparethe risk of female and male carriers (12). The similarity be-tween the results of studies of T1IEM carriers and humanD;G families emphasizes that mice with Robertsonian trans-locations are an important model for studying the behavior ofhuman translocation chromosomes.Our crosses of (TW/+ TA/+) F, hybrids with normal

AKR/J mice were done to determine if females had a higherfrequency of aneuploid progeny than males. However, noneof the crosses produced aneuploid fetuses. This finding wasunexpected because of the trisomic mouse detected in one F,cross and the meiotic studies of (TW/+ TA/+) F, males thatdemonstrated frequent nondisjunction of trivalents (Table 2).When more than two trivalents are present at M I, such as inthe F1 from crosses between Mus musculus X Mus poschiavinus(7 trivalents), more than 50% unbalanced M II complementsare observed, trisomic fetuses are found in backerosses, andfertility of the F1 is decreased (13).The single trisomic from our F1 crosses (TW/+ TA/+) 9

X (TW/+ TA/+) d' contrasts with the 12% incidence oftrisomy 19 at birth resulting from crosses between F1 miceheterozygous for both TlWh and T163H (6). In the lattercase, trisomy was due to regular nondisjunction of quad-rivalent chromosomes, while the present instance was relatedto trivalent nondisjunction.A total of seven mice (2 male, 5 female) homozygous for

both TW and TA with 36 chromosomes resulted from thecrosses shown in Table 1. Like homozygotes for a singleRobertsonian translocation with 38 chromosomes (1, 3) theywere phenotypically normal, their meiotic studies showedno evidence of nondisjunction, and their fertility was not re-duced. Evidently, the translocation chromosomes of thelarge ring bivalents of such homozygotes are comparable tothe chromosomes of normal autosomal bivalents in their con-sistently balanced mode of disjunction.While Robertsonian fusion has been considered one of sev-

eral possible evolutionary mechanisms, no obvious phenotypicalteration occurred in mice with the polymorphisms generatedby crossing (TW/+ TA/+) F1 hybrids. The studies ofRobertsonian polymorphism in the African pigmy-mouse byMatthey (14) demonstrated a similar phenomenon in the wild.The pigmy-mouse chromosome number varies from 18 to 34,while the number of chromosome arms remains 36. This sug-gests that further polymorphism could be developed in thelaboratory mouse by crossing the (TW/TW TA/TA) linewith other existing translocation stocks. While the doublestructure of the centromeric regions of the TW and TA trans-

locations indicated that paracentromeric heterochromatin isintact, there is no objective evidence that significant geneticchange occurs in Robertsonian fusion, which presumably re-quires breaks in the centromeric regions of both involvedchromosomes. Further comparison of characteristics of (TW/TW TA/TA) mice with their parent strains might reveal moreabout the effects of chromosomal fusion and their role in theevolutionary process.The quinacrine mustard- and Giesmsa-banding patterns

of the TW and TA chromosomes reported here are consistentwith those described by others. The quinarine mustard- andGiesmsa-bands are similar, indicating that either technique issufficient to identify specific mouse chromosomes. In additionto identifying linkage groups with certain chromosomes (num-ber 5, LG XIV; number 6, LG XI; number 9, LG II; number19, LG XII) (5), these techniques can be used to identifyspecific chromosomal aberrations during gestation and mayallow studies of the embryogenesis of anomalies associatedwith aneuploidy. Crosses of mice from existing translocationstocks can produce mice with various deviations from the nor-mal chromosome complement and can now be used as a sys-tem to study human chromosomal rearrangements.

1. Evans, E. P., Lyon, M. F. & Daglish, M. (1967) "A mousetranslocation giving a metacentric marker chromosome,"Cytogenetics 6, 105-119.

2. Leonard, A. & Deknudt, Gh. (1967) "A new marker forchromosome studies in the mouse," Nature 214, 504-505.

3. White, B. J. & Tjio, J. H. (1968) "A mouse translocationwith 38 and 39 chromosomes but normal N.F.," Hereditas58, 284-296.

4. Baranov, V. S. & Dyban, A. P. (1971) "A new markerRobertsonian translocation (centric fusion of autosomes)in the laboratory mouse Mus musculus," Cytologia 13,820-829.

5. Committee on Standardized Genetic Nomenclature forMice (1972) "Standard karyotype of the mouse, Musmusculus," J. Hered. 63, 69-72.

6. White, B. J., Tjio, J. H., Van de Water, L. C. & Crandall, C.(1972) " Trisomy for the smallest autosome of the mouse andidentification of the TlWh translocation chromosomes,"Cytogenetics, in press.

7. Nesbitt, M. & Francke, U. (1971) "Linkage groups II andXII of the mouse: Cytological localization by fluorochromestaining," Science 174, 60-62.

8. Miller, 0. J., Miller, D. A., Kouri, R. E., Allderdice, P. W.,Dev, V. G., Grewal, M. S. & Hutton, J. J. (1971) "Identi-fication of the mouse karyotype by quinacrine fluorescence,and tentative assignment of seven linkage groups," Proc.Nat. Acad. Sci. USA 68, 1530-1533.

9. Caspersson, T., Zech, L. & Johansson, C. (1970) "Dif-ferential binding of alkylating fluorochromes in human chro-mosomes," Exp. Cell Res. 60, 315-319.

10. Buckland, R. A., Evans, H. J. & Sumner, A. T. (1970)"Identifying mouse chromosomes with the ASG technique,"Exp. Cell Res. 69, 231-236.

11. Baranov, V. S. & Dyban, A. P. (1971) "Embryogenesis andpecularities of karyotype in mouse embryos with centricfusion of chromosomes (Robertsonian translocation),"Ontogenez 2, 164-176.

12. Hamerton, J. L. (1971) Human Cytogenetics (Academic PressNew York), Vol. 1, pp. 276-285.

13. Tettenborn, U. & Gropp, A. (1971) "Meiotic nondisjunc-tion in mice and mouse hybrids," Cytogenetics 9, 272-283.

14. Matthey, R. (1970) "L' 'eventail robertsonien' chez lesMus (Leggada) africains du groupe minutoides-musculoides,"Rev. Suisse Zool. 77, 625-629.

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