on a Macinotsh I1 ci (Apple Computer,. Inc.). Flow Karyotype Normalization. All of the swine bivariate flow karyotypes and the human-swine mixed karyotype ...
0 1992 Wiley-Liss, Inc.
Cytometry 13:703 -71 0 [ 1992)
Swine Chromosomal DNA Quantification by Bivariate Flow Karyotyping and Karyotype Interpretation’ Annette Schmitz2, Brigitte Chaput, Pierre Fouchet, Marie Noelle Guilly, Gerard Frelat, and Marcel Vaiman Laboratoire de Cytometrie, DPTE, DSV, Centre #Etudes Nucleaires, 92265 Fontenay aux Roses (A.S.,B.C., P.F., M.N.G., G.F.) and Laboratoire de Radiobiologie Appliquee, DPTE, DSV, Centre d’Etudes Nucleaires, 91191 Gif sur Yvette, (M.V.) France Received for publication December 13, 1991; accepted April 20, 1992
Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the “DNA line” in swine identical to the human “DNA line.” Estimation of the DNA content in megabase pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1,13, 6,5,10,16,11,18, and Y were assigned to
Flow cytogenetic techniques already contribute greatly to our knowledge of the genome in mammals. They have, for instance, proved invaluable in detecting numeral and structural aberrations associated with human genetic diseases. They have also been extensively applied to human chromosome sorting, thereby permitting the construction of chromosome-specific DNA libraries (see ref. 6 for review). Applied to domestic species, flow cytometric techniques have been particularly straightforward in swine cytogenetic analyses, as most of the chromosomes in this species are both fairly different in size, and are fewer in number compared with human or bovine chromosomes (22). Flow cytogenetics and chromosome sorting therefore appear especially relevant for the ongoing European PiGMaP project (2). Among the aims of the project is the construction of chromosome-specific libraries, in order to provide as quickly as possible chromosome-assigned markers, which in conjunction with other techniques will allow coverage of the whole genome. A prerequisite to production of chromosome-specific libraries is accurate assignment of all, or at least most,
peaks A, B, C, K, L, N, 0, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9,4, and X to peak H. CI 1992 Wiley-Liss, Inc. Key terms: DNA line, chromosome identification
of the swine chromosomes to the peaks of the flow karyotype and the estimation of the swine chromosomal DNA content. We therefore further developed studies on bivariate flow karyotypes in swine, as previous results showed that good chromosome resolution was easily achievable (7,s). All swine chromosome types could be resolved into individual peaks except two autosomal pairs plus chromosome X, which were confounded (15). The work reported here concerns the establishment of a “DNA line” in swine, a technique successfully applied to human chromosomes (17, 3, 18).The DNA line was used to estimate the chromosomal DNA content and to make the putative assignments for all chromo-
‘This work was supported hy the Commissariat B 1’Energie Atomique CEA FA 1123.03 and EEC BRIDGE “PiGMaP.” ’Address reprint requests to, Annette Schmitz, Laboratoire de Cytometrie, DPTE, DSV, Centre dEtudes Nucleaires, Bp 6,92265, Fontenay aux roses, France.
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somes to the various peaks on the bivariate flow karyotype.
MATERIALS AND METHODS Animals We analyzed the karyotypes of 11pigs of both sexes, 9 males and 2 females, of which 3 carried a t(3,7)(p1.3: q2.1) reciprocal translocation (5,7). Ten pigs, including the latter, were of the Large White breed, and the last was a Large White x Meishan F1 animal. Six pigs belonged to the same litter as the translocated pigs, whereas the other five were unrelated. Preparation and Culture of Lymphocytes From Peripheral Blood Sterile peripheral blood was withdrawn by puncture of the superior vena cava and collected on heparin. Mononuclear cells were isolated by centrifugation of 1:3 (v:v) phosphate-buffered saline (PBS) diluted blood on a Ficoll density solution (Pharmacia, Uppsala, Sweden) (specific gravity 1.077) at l,OOOg, for 25 min a t 18°C. After a single washing with PBS the cells were cultivated at 2.5 x lo6 cells/ml in 10 ml of MacCoy’s medium (Eurobio). The medium was supplemented with 10% autologous fresh plasma, 1%P-mercaptoethanol, and 0.2 ml phytohemagglutinin (PHA) (Eurobio; contents of a vial dissolved in 5 ml distilled water). Cell culture was carried out a t 37°C for 72 h (7). Cell Lines and Culture The swine lymphoblastoid sub-cell line L52, kindly provided by Dr. Kaeffer, was isolated from circulating mononuclear cells recovered from a miniature boar previously injected with porcine retrovirus-producing cells (9). The cell line was maintained in culture by conventional techniques in RPMI medium supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 2 mM glutamine, 10,000 IU/ml penicillin, 10 mg/ml streptomycin?and 50 pM P-mercaptoethanol. The human lymphoblastoid cell line Boleth, kindly supplied by Prof. D. Cohen, CEPH (1,211, was immortalized from a normal man using Epstein-Barr virus (EBV). The cell line was maintained in culture in RPMI medium supplemented with 10% FCS, 2.5 mM sodium pyruvate, 25 mM HEPES, 2.5 mM glutamine, 10,000 IUiml penicillin, and 10 mg/ml streptomycin. Chromosome Preparation Chromosomes were isolated from metaphasic cells, accumulated by a colcemid block of 21 h for short-term lymphocytes (1.5 pg/ml) and 5 h for the cell lines (0.1 pg/ml), according to Van den Engh et al. (19), and generally analyzed within 48 h. Briefly, cells were swollen in a low ionic strength buffer (50 mM KC1, 10 mM MgSO,, 5 mM Hepes, 3 mM D!M’, pH 8.0) and cell membranes were disrupted by the action of a mild detergent (0.25% Triton X-100) (Sigma). Chromosome suspensions were stained with the DNA dyes chromomycin A3 at 50 pgiml (Sigma) and Hoechst 33258 at
3.3 p,g/ml (Riedel de Haen, Seelze, FRG) 2 h before analysis. Sodium-citrate and sodium-sulphite were added to chromosomes 15-30 min before measurement ~ solution (100 mM Na-citrate, 250 from a 1 0 stock mM Na-sulphite) to improve karyotype resolution (20). Flow Cytometric Analysis Bivariate flow analysis of chromosome suspensions was performed with a standard dual beam flow sorter ATC 3000 (Odam Bruker, Wissenbourg, France) equipped with a Spectra Physics 2025 laser (Mountain View, CA) and a Coherent Innova 90.5 laser (Palo Alto, CA). The primary laser was tuned to emit the ultraviolet (358-361 nm) to excite Hoechst 33258 (0.4W), and the second laser was tuned to 458 nm to excite chromomycin A3 (0.4 W). Hoechst 33258 and chromomycin A3 fluorescence signals were separated with the appropriate spectral filter combination (Melles Griot, Irvine, CA) (4,lO). Flow data were accumulated as a list of 50,000 events. Monovariate flow analysis of chromosome suspensions stained by ethidium bromide was performed using the same flow cytometer with one laser beam tuned to 488 nm to excite ethidium bromide. Fluorescence and wide-angle light scatter signals were separated with the appropriate filter combination and the flow data accumulated as a list of 15,000 events. Flow Karyotype Analysis The peak volumes and the mean HO and CA3 fluorescence intensities of each chromosome population resolved on the bivariate flow karyotype (peak coordinates) were determined from the list mode data. The fluorescence offsets of the flow cytometer were calculated from swine bivariate flow karyotypes measured with and without neutral density filters. Neutral density filters were added before the Hoechst 33258 and chromomycin A3 fluorescence signal separation. Correlated plots of chromosome peak positions were realized. The fluorescence offsets were calculated and were equal to 22 channels for the chromomycin A3 and 8 for the Hoechst 33258 pathways. Corrections of the flow data for the fluorescence offsets, karyotype normalizations, and projections on the “DNA line,” as well as monovariate distribution modeling from the projection values, were obtained after transferring the peak coordinates and volumes determined from the flow data for each flow karyotype to the computer spreadsheet software Microsoft Excel (version 2.2 from Microsoft Corp.) on a Macinotsh I1 ci (Apple Computer, Inc.). Flow Karyotype Normalization All of the swine bivariate flow karyotypes and the human-swine mixed karyotype were normalized by setting the average HO and CA3 intensities (peak coordinates) of all the normal swine autosomes to 100 for each karyotype. The human karyotype was normalized to the swine karyotypes after determination of values
SWINE BIVARIATE FLOW KARYOTYPE INTERPRETATION
for HO and CA3 (102 and 86, respectively) corresponding in pig to 100 on the mixed karyotype.
Bivariate Flow Data Projection The distances D, on the DNA line were calculated by orthogonal projection for each resolved peak, n, on the bivariate flow karyotype, characterized by its peak coordinates HO, and CA3, D,
=
CA3, coscl
+ HO,
sinct
where Tana = HO, - HOJCA3, - CA3,, x and y being the peaks through which the line runs.
Monovariate Model Distribution A flow karyotype is produced typically by the measurement of 30,000 chromosomes. The fluorescence intensities are cumulated to produce a monovariate or bivariate distribution. Individual chromosome measurements are assumed to be normally distributed around mean values and can be approximated with Gauss functions. The shape of the individual chromosome peaks represents the error in the fluorescence measurement so that the parameter describing the peak shape will vary with the peak position (SD, = CV x D,,). Ideally, one mean peak position has to be determined for each chromosome and even for each homologue so that the flow karyotype can be properly modeled. As we were unable to resolve all the chromosomes into individual peaks, we considered the measured means as an average position of the chromosome types or homologues composing the peak. The volume of each peak was converted to a “frequency per cell basis” by normalizing to the total volume produced by all the chromosomes using the relationship:
705
We therefore studied in parallel human and swine chromosomes. The chromosomes were obtained from a human lymphoblastoid cell line immortalized from a normal individual (1,21) (Fig. 1A) and from a swine lymphoblastoid cell line presenting a normal karyotype according to flow analysis (15)(Fig. 1B). Humanswine mixed chromosome suspensions were obtained either from a mix of the two cell lines before chromosome isolation or from the chromosome suspensions prepared separately (Fig. 1C). The chromosome suspensions were stained with the Hoechst (HO) and chromomycin A3 (CA3) dyes and bivariate flow analyses were performed with the same apparatus settings. As shown in Figure l A , the human karyotype displayed the typical pattern corresponding to the normal karyotype, comprising 20 resolved peaks. The flow karyotype of the L52 swine lymphoblastoid cell line is displayed in Figure 1B. As not all swine chromosomes were assigned to the various peaks, we used the nomenclature proposed earlier, based on the peak contents (15). In brief, the various peaks denoted from A to Q and Y contained only one chromosome type, except for peak H, which was composed of two autosomal pairs plus X. In this experiment the 19 swine chromosomal pairs were resolved into 17 peaks since the peaks I and J were confounded. Figure 1C shows a flow analysis of a chromosome mixture from the two species. It displayed at least 33 peaks, indicating good discrimination between swine and human chromosomes. To allow fluorescence offset correction and karyotype normalization, the mean peak positions were calculated for the three flow measurements in Figure 1A-C. As described in Materials and Methods, the average HO and CA3 intensities were determined from the individual peak positions of the swine autosomes and were set to 100 in the swine and the mixed karyotype, allowing human chromosome normalization to swine karyotypes using the same method.
Volume of the test peak x 38 sum of all peak volumes A gaussian distribution was calculated for each proDNA Lines in Human and Swine jected peak position. The frequencies (F,) were calcuA line giving access to human DNA content by orlated over a range of 256 channels (C) with N, being taken as the number of chromosomes per cell compos- thogonal projection of the chromosomal peaks on the ing the peak n, and D, being the calculated distance for bivariate flow karyotype denoted DNA line, has been the peak n on the projection line. The CV was set at 2%. defined experimentally by Trask et al. (18). This line was chosen on the human flow karyotype by testing various projection angles. The same procedure was applied to the swine bivariate flow karyotype corrected The individual gaussian distributions corresponding to for the fluorescence offsets in our flow system and northe various resolved peaks were then summed to obtain malized. To test the swine DNA line we compared, for the monovariate model distribution. the same animal, the monovariate flow karyotype after ethidium bromide staining and the monovariate model RESULTS karyotype calculated after orthogonal projection of the Normalization of the Human and Swine bivariate flow karyotype, under various angles (0Bivariate Flow Karyotypes 90’). As shown in Figure 2, the experimental and calAn approach to determination of swine chromosomal culated distribution using the selected projection angle DNA content was to take the DNA content of human (44” on the normalized karyotype) showed good correchromosomes as standard, and to make use of the tool spondence while other tested angles did not improve designated “DNA line,” developed by Trask et al. (18) the results (data not shown). As no chromosomal peak for the analysis of the human bivariate flow karyotype. on the swine bivariate flow karyotype could be used to
N, =
DNA content from bivariate flow karyotype
0
Ethidiumbromide fluorescence intensity
256
FIG. 2. Correspondence, for the same swine, between the experimental and modeled monovariate chromosome distributions. A Model frequency distribution obtained by projecting the peak positions on the HO vs. CA3 karyotype onto the selected DNA line, calculating Gauss distributions for each projected peak position and summing the individual distributions. B: Monovariate flow karyotype of chromosomes stained with ethidium bromide. The fluorescence intensities were quantified using a single beam flow cytometer.
define the selected projection line through a given peak and the origin, we chose t o define the projection angle from a parallel line running through three peaks on the bivariate flow karyotype. The peaks H, P, and Q, well resolved, reasonably separated, and aligned, were selected and tested on the different swine karyotypes. A variation range of 3.5" was observed for the projection angle (Y defined by these three peaks, among a set of 11 animals. The HPQ line was therefore considered
FIG.1. Bivariate flow karyotype of mitotic chromosomes stained with Hoechst 33258 and chromomycin A3. Fluorescence intensities were quantified to obtain these three measurements using a dual beam flow cytometer with the same apparatus settings. The karyotypes are displayed as Hoechst 33258 fluorescence intensity versus chromomycin A3 fluorescence intensity cytograms. The cell lines were: (A) Boleth, a normal human lymphoblastoid cell line, (R) L52, a swine lymphoblastoid cell line, and (C) a mixture of A and B.
707
SWINE BIVARIATE FLOW KARYOTYPE INTERPRETATION I m
-y
= -0.86548
+ 1.4349~R= 0.99888
C
3
I 0 -
FIG.3. Peak positions after offset correction and normalization on the bivariate flow karyotypes presented in Figures 1A-C. The karyotypes were normalized to each other by setting the average HO and CA3 intensities of all swine autosomes to 100 in the swine and mixed flow measurement. The human karyotype was normalized in the same way after evaluation of the normalization values on the mixed karyotype. The chromosomes were from the human lymphoblastoid cell line, the DNA line running through the origin and peak of chromosome 4 (A) and from the swine lymphohlastoid cell line, a line running through peaks H, P, and Q (0).
provisionally as representative of the true swine DNA line throughout the experiment. We then compared the DNA line characterized in swine to the DNA lines defined in human. It was not possible to compare our results to those of Boschman et al. (31, as the DNA line defined by them was only characterized by an angle in a normalized system different from ours. Trask et al. (18) defined their human DNA line as running from the origin to chromosome 4. We could demonstrate that on the human-swine mixed flow karyotype the human DNA line running through human chromosome 4 and the origin was characterized by an angle very close to that of the swine DNA line and within the range of angle variation observed among the swine karyotypes (Fig. 3). To confirm the validity of the DNA line for the human chromosomes in our system, we calculated the distances on the DNA line of all the human chromosomes. These calculated distances were then correlated to the DNA content measurements of human chromosomes previously defined by Mayall et al. (13). A linear relation with a coefficient of correlation of 0.999 was found (data not shown).
Estimation of the DNA Content of Swine Chromosomes Having demonstrated both the relevance of the human DNA line in our system and its similarity with the swine's, we assumed that the dye interactions in human and swine were not significantly different. Thus, we felt justified to use the human chromosomal DNA
FIG. 4. The relationship between our estimates of chromosome DNA content made from the HO versus CA3 human flow karyotype by projection onto the DNA line and the average DNA content in megabase-pairs proposed by Trask et al. (17) from the karyotypes of 33 unrelated individuals.
content as standard to estimate that of the swine using the DNA line tool. The relation between the human relative DNA content measured by us and the average DNA content in megabase pairs proposed by Trask et al. (17) from the karyotypes of 33 unrelated individuals is described by: Y
=
1.43X - 0.9
(1)
where Y is the DNA content in megabase pairs and X the peak position on the DNA line. The coefficient of correlation was 0.999 (Fig. 4). This relation was applied to the corrected and normalized flow karyotypes of 11 swine, among which 3 carried a t(3,7) translocation. Only the normal chromosomes were taken into account. The individual distances of each peak from the 11 karyotypes on the DNA line were averaged and the standard deviations were calculated. This gave the estimate of the DNA content in megabase pairs of all the swine chromosomes. The results are listed in Table 1.
Chromosome Assignment to Peaks The average relative DNA contents of the chromosomes of the 11 swine evaluated above were compared t o the average swine chromosome length on metaphase spreads measured previously by Lin and coworkers (12). A linear relationship was obtained between the relative distances on the DNA line and the average chromosome size (Fig. 5). From this relation we suggest the provisional assignment of chromosomes 1, 13, 6 , 5 , 10,16,11,18,andY to peaks A, B, C, K, L, N, 0, Q , and Y, respectively. Peaks D and E, which were very close and even confounded on half the bivariate flow karyotypes, were assumed to contain chromosomes 2 and 14. Although peaks P and M were resolved on all karyotypes, they displayed close DNA contents and were assumed to correspond to chromosomes 17 and 12, though
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Table 1 Chromosomal DNA Content Estimation
Peak A B
C
D E
F G
H1 H2 H3
I J K L M N 0 P
Q Y
DNA quantification from 11 swine bivariate flow karyotypes" Mean peak position Standard deviation" (mean D,) (SD,) 206 2.4 161 2.0 124 1.7 118 0.5 118 0.4 104 3.3 98 2.2 102 0.5 102 1.2 101 0.7 112 2.1 111 2.0 83 0.7 72 0.8 52 1.9 71 1.7 66 2.1 54 1.0 48 1.3 33 2.1
Estimation of the DNA contentb DNA contentihaploid genome Fraction of the male genome (%) (C,) 2 x C,/[SC, x 2-(Cx + Cy)l x 100 295 5.5 230 4.3 177 3.3 168 3.1 168 3.2 149 2.8 141 2.6 145 2.7 146 2.7 145 2.7 161 3.0 3.0 158 118 2.2 103 1.9 74 1.4 101 1.9 94 1.8 77 1.4 68 1.3 47 0.9
"The DNA was quantified by projection of the peaks from bivariate flow karyotypes of 11 swine onto the DNA line. "The DNA content in Mbps was calculated using DNA quantification by flow cytometry and the relation defined from human (Eq. 1). "Standard deviations calculated from the data of 11 swine.
-5. -
-y = 0.30317 + 0.046522~R= 0.99716
H, respectively (151, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, but without a specific assignment, and chromosomes 9,4,and X to peak H. The results are summarized in Table 2.
DISCUSSION In this study we devised the DNA line on the swine bivariate flow karyotype aiming to define both swine chromosome DNA content as well as the assignment of most swine chromosomes to specific flow peaks. The DNA line concept was first proposed by Trask et al. (18) for comparison and quantification of human chromosomal DNA content. Comparisons showed good agreement between the values found for relative chromo0 1 I I I somal DNA content determined either by CYDAC 0 64 128 192 256 DNA content from bivariate flow karyotypes measurements, by monovariate flow analysis, or from bivariate flow karyotypes using the DNA line method FIG.5. Relationship between swine chromosome DNA content es- according to Trask et al. and Boschman et al. (3,17,18). timates made from HO vs. CA3 flow karyotypes and estimates of relative chromosome length on metaphase spreads (12). Flow esti- Nevertheless, some discrepancies between the cited aumates of DNA content represent the mean measured for each chro- thors about the absolute position of the DNA line were mosome type in flow karyotypes of 11 different swines. The karyo- emphasized by Boschman et al. (3), although the diftypes were corrected for the offset and normalized to each other by ference between the angles of the two DNA lines (4.1") setting the average HO and CA3 intensities of all autosomes to 100. was found to result in an average difference in relative The DNA content was determined from the projection of each chrochromosomal DNA content of about 1%.These obsermosome type on the DNA line. vations suggest that a more appropriate definition of the DNA line may be needed, although in our case, a it was hazardous a t this stage to decide which (12 or 17) slight imprecision of the projection angle can be tolercorresponded to which peak. Of the remaining chromo- ated as the derived data are to be used for comparison somes ( 3 , 7, X, 8, 15, 9, and 4),chromosomes 3, 7, and to chromosomal length and for a first estimation of the X, which were assigned previously to peaks F, G, and swine chromosomal DNA content. I
SWINE BIVARIATE FLOW KARYOTYPE INTERPRETATION
709
swine chromosomal DNA content are important for establishing cloning strategies (16) and for the rescaling of physical and composite maps (14). The relation between the chromosome length and the Flow peak Chromosome relative peak position on the DNA line permitted assignment of all chromosomes to specific peaks. All peaks comprised one chromosome type, except for peak H, which is composed of three chromosome types, namely chromosomes 9, 4,and X. The assignments of chromosomes 3 and 7 to peaks F and G have been established previously by polymerase chain reaction (PCR)on sorted chromosomes and study of translocated t(3,7) pigs. Chromosomes 1 and 13 were assigned to peaks A and B, and chromosomes X and Y to peaks H and Y, respectively (15). These results were incorporated into the peak content estimation we propose. In conclusion, this report is a first attempt to provide a general chromosomal assignment in swine. This method, of combining the use of the DNA line with comparison to chromosomal length, constitutes a general strategy that could be applied to other species, especially when chromosome distribution prediction based on standard metaphase idiograms (11) is inapplicable as for swine (Schmitz et al., unpublished results). Complete assignment by molecular biology 0 11 techniques, such as PCR on flow-sorted chromosomes, would have been laborious, as each chromosome-specific probe has to be tested on all the flow peaks. Moreover, so far, only about 50 sequences have been mapped in swine and we do not yet possess an assigned known sequence on each chromosome. Estimation of the chromosome localization on the swine bivariate flow karyotype is thus invaluable to speed up the subsequent confirmation by methods taking advantage of chromosome sorting and hybridization with chromosome-specific probes. The results have already been used successfully After correction for the fluorescence offsets in our to sort specific chromosomes for the construction of flow system, our results with human chromosomes chromosome-specific libraries in accordance with the were in agreement with the data of Trask et al. (18). PiGMaP project. The DNA contents calculated using the DNA line runACKNOWLEDGMENTS ning through the origin and chromosome 4 fitted both We thank Dr. Bertrand Kaeffer, INRA, Laboratoire de Pathe values measured by CYDAC and the estimations drawn from the analyses of karyotypes of 33 unrelated thologie Porcine et Immunologie, Nouzilly, France, for proindividuals. These results show that a DNA line de- viding the L52 swine lymphoblastoid cell line and J.C. Gaufined by internal references such as chromosomal cher for fruitful discussions concerning the offset problem. peaks on the flow karyotype may be transposed from LITERATURE CITED one flow system to another. Thus we preferred to define 1. Albertsen H, Abderrahim H, Cann H, Dausset J, Le Paslier D, the DNA line in swine as running through three peaks Cohen D: Construction and characterisation of a yeast artificial rather than by an angle. In a system where human and chromosome library containing seven haploid human genome equivalents. Proc Natl Acad Sci USA 87:4256-4260, 1990. swine bivariate flow karyotypes are normalized, the DNA line defined experimentally on the swine bivari- 2. Archibald A, Haley CS, Anderson L, Bosma AA, Davis W, Fredholm M, Geldermann H, Gellin J, Groenen M, Gustavsson I, 01ate flow karyotype and the human DNA line running livier L, Tucker EM, van de Weghe A: PiGMaP: A European through the origin and the human chromosome 4 are initiative to map the porcine genome. h i m Genet 22(Suppl 1): characterized by the same angle. Thus we considered 82-83,1991. that the dye interactions in human and swine chromo- 3. Boschman G, Rens W, van Oven C, Manders E, Aten J: Bivariate flow karyotyping of human chromosomes: Evaluation of variasomes were not significantly different. Accordingly, we tion in Hoechst 33258 fluorescence, chromomycin A3 fluoresfelt justified to assess the swine chromosomal DNA uscence, and relative chromosomal DNA content. Cytornetry 12: ing the human chromosomal DNA content as standard. 559-569, 1991. As for the human project, physical parameters such as 4. Delattre 0, Grunwald M, Bernard A, Grunwald D, Thomas G, Table 2 Chromosome Assignment to Peaks
EF=l
710
5.
6. 7. 8. 9.
10. 11. 12. 13.
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Frelat G, Aurias A: Recurrent t(11;22) breakpoint mapping by chromosome flow sorting and spot blot hybridization. Hum Genet 78~140-143, 1988. Gabriel-Robez 0, Jaafar H, Ratomponirina C, Bosher J, Bonneau J, Popescu CP, Rumpler Y: Heterosynapsis in a heterozygous fertile boar carrier of a 3;7 translocation. Chromosoma 97:26-32, 1988. Gray JW (ed): Flow Cytogenetics. Academic Press, Inc., New York, 1989. Grunwald D, Geffrotin C, Chardon P, Frelat G, Vaiman M: Swine chromosomes: Flow sorting and spot blot hybridization. Cytometry 7:582-588, 1986. Grunwald D, Frelat G, Vaiman M: Animal flow cytogenetics. In: Flow Cytometry: Advanced Research and Clinical Applications, vol. 1, Yen A (ed). CRC Press, Inc., Boca Raton, 1989, pp 132-140. Kaeffer B, Bottreau E, Phan Thanh L, Olivier M, Salmon H Histocompatible miniature boar model: Selection of transformed cell lines of B and T lineages producing retrovirus. Int J Cancer 46:481-488, 1990. Langlois RG, Yu LC, Gray JW, Carrano AV: Quantitative karyotyping of human chromosomes by dual beam flow cytometry. Proc Natl Acad Sci USA 79:7876-7880, 1982. Lebo RV, Golbus MS, Cheuny MC: Detecting abnormal human chromosome constitutions by dual laser flow cytogenetics. Am J Med Genet 25519-529, 1986. Lin C, Biederman B, Jamro H, Hawthorne A, Church R Porcine (Sus scrofu dornestica) chromosome identification and suggested nomenclature. Can J Genet Cytol22:103-116, 1980. Mayall B, Carrano A, Moore I1 D, Ashworth L, Bennet D, Mendelsohn M: The DNA-based human karyotype. Cytometry 5376385, 1984.
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Sci USA 88:7474-7476, 1991. 15. Schmitz A, Chardon P, Gainche I, Chaput B, Guilly MN, Frelat G, Vaiman M: Pig standard bivariate flow karyotype and peak assignment for chromosomes X, Y, 3 and 7. (in press). 16. Southern EM: Applications of DNA analysis to mapping the human genome. Cytogenet Cell Genet 32:52-57, 1982. 17. Trask B, van den Engh G, Mayall B, Gray JW: Chromosome heteromorphism quantified by high resolution bivariate flow karyotyping. Am J Hum Genet 45:739-752, 1989. 18. Trask B, van den Engh G, Nussbaum R, Schwatz C, Gray J: Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping Cytometry 11:184-195, 1990. 19. van den Engh G, Trask B, Cram S, Bartholdi M: Preparation of chromosome suspensions for flow cytometry. Cytometry 5:108117, 1984. 20. van den Engh G, Trask B, Lansdorp P, Gray J : Improved resolution of flow cytometric measurements of Hoechst- and chromomycin-A3-stained human chromosomes after addition of citrate and sulfite. Cytometry 9:266-270, 1988. 21. Yang SY, Milford E, Hammerling U, Dupont B: Description of the reference panel of B-lymphoblastoid cell lines for factors of the HLA system: The B-cell line panel designed for the Tenth International Histocompatibility Workshop. In: Immunobiology of HLA, vol. 1,Dupont B (ed). Springer-Verlag, New York, 1989, pp 11-19. 22. Yerle M, Galman 0, Echard G The high resolution GTG-banding pattern of pig chromosomes. Cytogenet Cell Genet 56:45-47, 1991.
