Chimaeric and Constitutive DNA Fingerprints in the

PRIMATES, 41(1): 49-61, January 2000 49

Chimaeric and Constitutive DNA Fingerprints in the Common Marmoset (Callithrixjacchus)

E. N. SIGNER, University of Leicester G. ANZENBERGER, Universitiit Ziirich-lrchel

and A. J. JEFFREYS, University of Leicester

ABSTRACT. Marmosets normally produce dizygotic twins sharing placental blood vessels and exchang- ing bone marrow cells. Each individual is therefore likely to be a blood chimaera. To date, marmosets had only been DNA fingerprinted using blood samples and probes 33.6 and 33.15, resulting in highly similar fingerprints among litter mates and little variation between other individuals, thereby limiting this method's use for individual identification and parentage testing. In this study, novel probes were applied to detect greater polymorphism and to produce individual-specific DNA fingerprints. As expected, blood DNA profiles of twins and triplets were virtually identical, confirming chimaerism in this tissue and identifying litter mates. Furthermore, these profiles were sufficiently variable to distinguish between sibs from different litters and between all other individuals. To produce individual-specific DNA fingerprints, the use of DNA extracted from tissues poor in leukocytes was essential. The findings demonstrate that, despite extensive blood chimaerism, marmoset colonies can be effectively DNA fingerprinted for individual identification, zygosity testing, and relationship studies.

Key Words: DNA fingerprinting; Marmosets; Blood chimaerism; Constitutive DNA.

INTRODUCTION

Common marmosets (Callithrix jacchus), like other marmosets and tamarins, are peculiar among primates. As a rule, they produce two non-identical infants at a time (HILL, 1926) which share blood vessels through extensive placental anastomoses in early embryonic life (WISLOCKI, 1939). Consequently, haematopoietic and lymphatic cells are exchanged, giving rise to bone marrow chimaeras (BENIRSCHKE et al., 1962).

DNA fingerprinting, first developed for humans, allows for the simultaneous screening of multiple hypervariable minisatellite loci present in the genome (JEEFREYS et al., 1985). Inde- pendently of an individual's nucleated cell type, the resulting DNA profiles are - - with rare exceptions - - individual-specific, somatically stable and inherited in a Mendelian fashion. Conveniently, the technique works in almost all vertebrates, and sufficient DNA for typing can be extracted from small blood samples. Major applications in animals include identification of individuals and inbred lines, pedigree testing and monitoring genetic variability in populations and migration between them (reviewed in PENA et al., 1993). Usually, sufficient polymorphisms are detected with the commonly used human probes 33.15 and 33.6. However, in some species hardly any polymorphism is observed, for example in cheetahs (MENOTTI-RAYMOND & O'BRIEN, 1993), badgers (T. BURKE, pers. comm.), naked mole rats (FAULKES et al., 1997), and beavers (ELLEGREN et al., 1993), presumably due to severe population "bottlenecks," inbreeding or an intrinsic lack of variability at the loci detected. Relatively restricted DNA fingerprint variation has also been reported for the common marmoset, in both captive and wild colonies

50 E.N. SIGNER et al.

(DIXSON et al., 1988, 1992). In this case, the species' characteristics of blood chimaerism (diminishing constitutive DNA differences between full sibs) and living in extended family groups (greater chance of sampling close relatives) have been implicated in causing the high level of DNA fingerprint similarity seen.

In this study, we employed novel probes and used DNA from various tissues in addition to blood to generate DNA fingerprints from a captive colony of common marmosets. Our goals were: (1) to achieve more variable DNA fingerprints from blood; (2) to produce constitutive and individual-specific DNA fingerprints by using tissue extracts; and (3) to investigate the extent of chimaerism.

MATERIALS AND METHODS

ANIMALS

Twenty-five animals were analyzed from a captive colony kept at the Psychological Institute, University of Ziirich (Table 1). They included one extended family with pedigree (Fig. 1), one breeding pair (animals 9 and 1 0 ) with one of their offspring (11 ) , an unlike-sex twin pair ( 1 A / 1 B ) and an adult female imported from Brazil ( 1 6 ) . Sampling was done over seven years on an opportunistic basis. Blood samples ( 1 - 2 ml) were drawn from femoral veins of anaes- thetized animals. Tissue samples were taken from animals that had died at birth or had to be euthanized for reasons of bad health. All samples were transferred into EDTA-coated tubes, frozen in dry ice and stored at - 80~ Table 1 gives details of the samples analyzed.

DNA FINGERPRINTING

High molecular weight DNA was extracted by proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. DNA concentration was either estimated by eye on 0.6% test gels or measured by fluorometry (Hoefer Scientific). After incubation with restriction endonucleases A l u I , H a e l I I , or H i n f I , 2/xg fragmented DNA per individual together with size marker DNA ( H i n d l I I digested ~, phage DNA or 1 kb DNA ladder, Gibco, BRL) were elec- trophoresed on 0.8% uniform agarose gels or 0.8%/1.5% composite gels (SIGNER et al., 1988) in

Table 1. Animals and DNA sources.

Animals Blood Animals Blood Spleen Liver K i d n e y Gonad Brain 4A + 1A* + + + + + 4B + IB* + + + + + 5A + 2A + + + + + 5B + 2B + + + + + 5C + 3A + + 6A + 3B + + 6B + 9* + + + + 7A + 10" + + + + 7B + 11" + + + 8A + 12 + + + + 8B + 13 + + + + + 1 4 + 16" + + 15 +

+ +

Individuals with the same number are litter mates. Numbers in bold indicate founder animals. * individuals not part of the pedigree in Figure 1.

DNA Fingerprints of Common Marmosets 51

Fig. 1. Pedigree of the extended family group analyzed. Males are represented by squares, females by cir- cles. Founder animals are in bold. ns: Individual not sampled.

TBE buffer (89 mM Tris borate, pH 8.2, 2.5 mM EDTA) until fragments smaller than 2 kb had run off the gel. After denaturation, the DNA was blotted onto a nylon membrane (Hybond NfP, Amersham) by capillary transfer. Prehybridization of the blots was done in 0.5 M phosphate, pH 7.2, 7% SDS, 1 mM EDTA at 65~ for six hours followed by hybridization overnight in fresh solution supplemented with 32p-labelled probe. After two washings for 10 min each in 2x SSC, 0.1% SDS at 65~ the blots were exposed at - 7 0 ~ for up to seven days with intensify- ing screens. The autoradiographs were analyzed by eye. DNA fingerprints on the same blot were compared and only well-resolved polymorphic bands scored. Bands within an elec- trophoretic distance of +0.5 mm and similar intensity were assumed to be identical. Bandsharing between unrelated individuals was calculated as 2nxy/(nx + ny), where nxy = number of bands shared by two animals, and nx, ny = total number of bands scored in animal x and y, respectively.

HYBRIDIZATION PROBES

Ten minisatellite probes were individually labelled with 32p [FEINBERG and VOGELSTE1N (1983) for DNA labelling; CARTER et al. (1989) for RNA labelling] and sequentially hybridized with genomic DNA digests on Southern blots: human DNA probes M S I , MS31, MS32, MS40 (WONG et al., 1987), MS51, MS228A (ARMOUR et ai., 1989) and pXg3 (WONG et al., 1986), pig DNA probe S0322 (SIGNER et ai., 1996), and RNA transcripts of human probes 33.15 and 33.6 (JEFFREYS et al., 1985).

Table 2. Average number of bands per individual and bandsharing in marmoset blood DNA profiles.

No. of animals No. of bands Average No. of Average Probe/RE compared scored ( k b * ) b a n d s / a n i m a l bandsharing 33.6RNA/Ahd 2 40 (>2.3) 28.0 0.57 33.15RNA/HaeIII 3 34 (>4.5) 20.0 0.54 MS3 l/Alul 2 40 (>4.0) 29.0 0.62 MS32/Alul 3 24 (>2.2) 13.0 0.44 MS40/AIul 3 38 (>2.3) 20.0 0.47 MS5 llAlul 2 44 (>4.0) 26.5 0.34 MS228A/Alul 3 21 (>2.0) 12.3 0.56 pXg3/Haelll 6 36 (>6.0) 14.5 0.48 S0322/Alul 3 21 (>3.0) 12.7 0.53

RE: Restriction endonuclease; *>kb values give size in kilobases above which the number of polymorphic bands indicated were scored.


MIXED DNA TESTING

For two twin sets (2A/2B, 3A/3B), five mixed gonadal DNA samples were produced as fol- lows: DNA from each twin was digested with AluI and concentration measured by fluorometry. DNA of twin A was then added to DNA of twin B, giving a series of mixed samples containing 5%, 10%, 15%, 20%, and 50% of twin A DNA in twin B DNA. Electrophoresis, blotting and hybridization (with probe S0322) was done as described.

RESULTS

BLOOD AND TISSUE DNA FINGERPRINTS

Extensive hybridization was observed between all human or porcine minisatellite probes and marmoset DNA. Each probe essentially detected a distinct set of polymorphic DNA fragments with only minor overlap. Nine probes were applied to blood samples from two to six unrelated animals per probe, yielding a mean number of polymorphic bands per individual of 12.3-29.0 and average bandsharing values of 0.34-0.62 (Table 2). Whereas co-born sibs (six twin pairs and one set of triplets; derived from four different sets of parents) consistently showed virtually identical blood profiles, all other individuals had distinct profiles. Examples of blood DNA profiles produced from presumably unrelated animals and a pedigree are shown in Figures 2 and 3, respectively. Furthermore, in the parental profiles (Fig. 3) several maternal- and paternal-specific bands were seen segregating in the offspring and each offspring's band could be traced back to either the father or the mother or both.

For some animals, DNA fingerprints were also produced from spleen, liver, kidney, gonad, and brain (Table 1). In these cases, AluI or HaelII were used which, in contrast to Hinfl, are not blocked by CpG methylation. Therefore any profile variation seen reflects differences in DNA sequence and not methylation that might vary between different tissues (JEFFREYS, 1987). In contrast to blood DNA profiles, tissue DNA fingerprints produced with any probe showed discriminatory bands even between twins (three pairs analyzed), and the less leukocytes a tissue contained the more differences were seen. For instance, in the female/male twin pair 1A/1B (Fig. 4a) at least six individual-specific bands were detected in kidney and liver DNA but none in spleen and blood, which instead showed profiles equivalent to a mixture of the two individuals' kidney or liver DNA fingerprints. Although spleen and blood profiles were very similar, greater band intensity differences between twins were seen in spleen, reflecting the presence of constitutive DNA superimposed by non-constitutive lymphocyte DNA. Similarly, in male twins 2A/2B (Fig. 4b, left panel), individual-specific bands could only be detected in the leukocyte- poor testis, liver and kidney. In addition, Figure 4b shows the different DNA fingerprints produced for this twin pair, using probes S0322 and 33.15RNA, which detect three and nine discriminatory fragments, respectively.

Differences in band position or intensity were also detected within single individuals between blood and tissue DNA profiles, indicating chimaerism in leukocyte-rich tissues (Fig. 5). Apart from female 10 (Fig. 5, right panel) which had identical DNA fingerprints irrespective of DNA source or probe used, all other nine individuals analyzed had distinct DNA profiles in blood and spleen compared with gonad, liver or kidney, suggesting that these animals, too, had fraternal twin (or triplet) partners and had exchanged blood cells. Chimaeric DNA profiles also differed from constitutive ones by containing bands in addition to the constitutive profile (nine animals scored, data not shown).


Fig. 2. DNA profiles produced from presumably unrelated individuals. Note the substantial variation seen between these individuals. DNA was isolated from blood, except for animal 12 from which spleen was used. Sizes of marker fragments are indicated in kilobases (kb).

As shown in Table 3, equivalent differences between twin partners were not identified by all probes. For instance 33.6RNA, 33.15RNA, MS 1, MS40, MS51, and S0322 detected differences in both band position and intensity in twin 2A but no differences or only intensity variation in twin 2B. Conversely, probe MS228A detected different band positions and intensities in twin 2B but only intensity differences in twin 2.4. Moreover, the probes were not equally sensitive at revealing chimaerism; however, when combining their results, they were highly informative for each animal. Interestingly, no evidence for chimaerism was found for female 10 with any of the six probes applied. Considering the overall performance of these probes, the joint probability of failure to reveal chimaerism was low (2x 10-3), rendering animal 10 a rare exception.

To summarize, blood DNA profiles were consistently specific to co-born sibs and only differed between other relatives or unrelated animals. Each probe produced individual-specific

54 E . N . SIGNER et al.

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constitutive DNA fingerprints from liver, kidney, gonad or brain DNA extracts. All like-sex twins tested were dizygotic. Leuko-chimaerism in blood was inferred from identical blood profiles of unlike-sex twins (five sets analyzed), and was also observed for single individuals showing differences between DNA profiles from blood or spleen versus other tissues (nine of ten animals investigated).

MIXED DNA TESTING

To estimate the level of chimaerism detectable by DNA fingerprinting, mixed DNA testing was performed using probe S0322 (Fig. 6). Based on the most prominent band (top arrow on the


Fig. 5. Detection of chimaerism within individuals. Chimaerism is inferred from differences in band position and band intensity (marked by arrows) between leukocyte-rich and leukocyte-poor tissues. Individual 10 shows no blood vs constitutive DNA differences.

right) discriminating twin 3A from twin 3B, twin 3A DNA admixture in twin 3B DNA was seen down to the 5% level. With respect to a fainter band again specific to twin 3A (bottom arrow), admixture was detectable down to 10%. In twin set 2A/2B two bands differed, one of which was prominent in twin 2A (bottom arrow on the left) and was visible in a DNA admixture of at least 15%. The fainter band (top arrow) was not informative (it would however be informative in a complementary test in which DNA of twin 2B is admixed to that of 2A). Since the intensity of the prominent band at the 15% level of admixture corresponded to that in both twins' blood profiles, it appeared that approximately 85% of the leukocytes in the blood of these twins have derived from twin 2B.


DISCUSSION

The human DNA fingerprinting system using blood and probes 33.6 and 33.15 previously revealed modest variability in the common marmoset (DtxsoN et al., 1988, 1992). By applying multiple novel DNA probes, we detected substantially greater polymorphism. Overall bandsharing was higher than in humans, suggesting less genetic variability in the marmosets. Yet, blood DNA typing could distinguish individual animals - - except twins or triplets - - and identify parental-specific DNA fragments segregating in the offspring. Within all six twin pairs and one set of triplets analyzed we found virtually identical blood patterns, indicating uniform chimaerism in leukocytes of co-born animals. These findings are in line with the regular twinning and haematopoietic chimaerism known for the species.

Assuming that chimaerism is restricted to haematopoietic cell lineages, we used DNA extracts from leukocyte-poor tissues to produce profiles of an individual's true genotype. Such constitutive DNA fingerprints revealed greater inter-individual differences than DNA profiles from blood and enabled us to distinguish twin partners in both twin pairs analyzed. For the first time, we demonstrate from the DNA that like-sex marmoset twins are dizygotic despite their identical blood DNA profiles. This is in sharp contrast to humans, where identical DNA fingerprints in the blood of twins are diagnostic for monozygosity (HILL & JEFFREYS, 1985).

In a mixed DNA experiment we were able to detect DNA admixture of as little as 5%. By comparing profiles from a series of mixed constitutive DNA of twins with that from their blood, we found a heavily skewed ratio of blood chimaerism. Distorted ratios had also been observed in cattle (PLANTE et al., 1992) and human (TIPPET'r, 1983) blood chimaeras analyzed by karyotyping lymphocytes, and perhaps reflects a different timing of blood cell and placental vessel formation between the co-developing embryos.

On the basis of variations seen in an individual's DNA profiles produced from leukocyte-rich and leukocyte-poor tissues, we were able to detect haematopoietic chimaerism in nine of ten animals. Even though the probes were not equally sensitive in detecting such differences, jointly they were highly effective. The ability to detect chimaerism depended mainly on the degree of difference between the twin partners' constitutive DNA and the extent of mutual blood cell exchange between them. Discriminatory bands of high intensity served as most sensitive markers. We identified one exceptional twin (female 10) showing no indication of chimaerism. Since her twin sib was not available for sampling, we could not determine whether this was a rare incidence of monozygotic twinning or, more likely, a case of extremely unequal chimaerism with her genotype dominating to more than 95% in her blood. Such highly unequal


Fig. 6. Mixed DNA testing. In two experiments constitutive (gonadal) DNA of twins was mixed at defined ratios to estimate extent of chimaerism (12 lanes on the left), and to determine the sensitivity of DNA fingerprinting for detecting DNA admixture (7 lanes on the right). Percentage values indicate the amount of twin A DNA added to twin B DNA. By comparing the intensity of the prominent band in twin 2B's constitutive DNA fingerprint (bottom arrow on the left) with that in each admixed DNA profile and the blood profile, an estimated blood DNA chimaerism of approximately 15% 2A DNA plus 85% 2B DNA was deduced. Since the blood profiles of these twins were indistinguishable, only one of them (2A) is shown and the above ratio of blood chimaerism applies to both animals. Chimaerism in spleen was about 20% in twin 2A but barely detectable in twin 2B. With respect to the two discriminatory bands in twins 3A/3B, DNA fingerprinting sensitivity was estimated at 5% for the intense band (top arrow) and 10% for the fainter band (bottom arrow).

chimaerism in marmoset lymphocytes is, indeed, known from chromosomal studies (ARDITO et al., 1995).

We also investigated the possibili ty that chimaerism might extend to non-haematopoietic tissues. It is thought that pluripotent endothelial cells lining blood vessels and primordial germline cells migrating from the yolk sac wall to the genital r idges might incidentally enter the twins' shared placental vessels and be exchanged (GENGONZIAN et al., 1980). We found no evidence


for chimaerism in liver, kidney, gonad or brain DNA extracts. Bearing in mind the sensitivity of DNA fingerprinting, chimaerism in these organs is unlikely to be higher than 5%. Assuming chimaerism is restricted to bone marrow cells, then any tissue with high leukocyte content would show chimaeric blood-like DNA profiles. We used spleen as an example, which con- firmed this expectation. To date, somatic non-haematopoietic chimaerism has been demon- strated by karyotyping of cultured cells in rare incidences of such twins in cattle (STRANZlNGER et al., 1981; PLANTE et al., 1992) and humans (GILGENKRANTZ et al., 1981; FARBER et al., 1989), but this has not been investigated as yet in marmosets. Even though apparently XX primary spermatocytes were reported in males of unlike-sex twins in marmosets (HAMPTON, 1973), they were suspected of being artifacts by FORD and EVANS (1977) later on. More recently, GENGOZIAN et al. (1980) provided evidence for the absence of functional germline chimaerism. This conclusion is in line with our finding in a separate study (unpubl. work), where we identified six different non-constitutive fragments in the parental blood DNA fingerprints - - acquired from their twin partners - - none of which was inherited by any of the two offspring. Further work using more sensitive means to detect DNA admixtures is needed to determine whether and in which cell lineages trace chimaerism occurs.

As a consequence of leukocyte chimaerism, blood DNA showed more fragments than constitutive DNA. Therefore, the average number of bands per individual and bandsharing will have to be determined from constitutive DNA in order to determine the true extent of DNA fingerprint polymorphism for the species. A major drawback is the need to use internal organs to get sufficient DNA for producing individual-specific DNA fingerprints. Currently, we are investi- gating alternative, less invasive sampling coupled with more sensitive PCR-based typing sys- tems.

CONCLUSIONS

In the common marmoset, leukocyte-rich tissues produce chimaeric DNA profiles which are not individual-specific but specific to litter mates and therefore suitable for identifying co-born offspring.

In contrast, leukocyte-poor tissues produce constitutive and unique DNA fingerprints useful for individual identification, twin zygosity determination and parentage testing.

If at all present, non-haematopoietic chimaerism is low and undetectable by DNA fingerprinting, and leukocyte DNA co-extracted in crude preparations from non-lymphoid organs does not interfere with the production of constitutive DNA fingerprints.

Acknowledgements. We are most grateful to Drs. THOMAS HEINZELLER, HANS-PETER LIPP, and CHRIS PRYCE for their invaluable help in blood and tissue sampling. We also wish to thank Dr. ROBERT D. MARTIN for constructive comments on the manuscript. This study was partially funded by grants to A. J. JEFFREYS from the Royal Society, Wellcome Trust and the Medical Research Council. The human minisatellite probes are the subject of Patent Applications. Commercial inquiries should be addressed to Cellmark Diagnostics, 8 Blacklands Way, Abingdon, Oxon OX14, UK.

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- - Received: December 24, 1998; Accepted: September 4, 1999

Authors' Names and Addresses: E. N. SIGNER, Department of Genetics, University of Leicester, Leicester LEI 7RH, England. e-mail: [email protected]; G. ANZENBERGER, Anthropologisches lnstitut, Universitiit Ziirich-lrchel, 8057, Ziirich, Switzerland; A. J. JEFFREYS, Department of Genetics, University of Leicester, Leicester LE1 7RH, England.

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Chimaeric and Constitutive DNA Fingerprints in the