Department of Microbiology and Molecular Genetics, College of Medicine, University of California,. Irvine, California 92717. Received 22 ... 10-6 and contained mostly single, but occasionally multiple, copies of the plasmid sequences. ...... Isozymes Curr. Top. Biol. Med. Res. 2:101-158. 28. Southern, E. M. 1975. Detection ...
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1985, p. 140-146 0270-7306/85/010140-07$02.00/0 Copyright © 1985, American Society for Microbiology
Vol. 5, No. 1
Selective Transfer of Individual Human Chromosomes to Recipient Cells PAUL J. SAXON, ERI S. SRIVATSAN, G. VICTOR LEIPZIG, JEANNE H. SAMESHIMA, AND ERIC J. STANBRIDGE* Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92717 Received 22 June 1984/Accepted 22 October 1984
Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt). Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca. 10-6 and contained mostly single, but occasionally multiple, copies of the plasmid sequences. These transformants actively express the Eco gpt marker. Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells. The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization. This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background. The complete library will represent the entire human karyotype.
ducts of genes on the remaining chromosomes has, for the most part, not been attained. Single chromosomes can be transferred to recipient cells via microcell-mediated chromosome transfer techniques (6, 7, 18). However, the problems of selection of the microcell hybrid and retention of the single human chromosome are, in principle, the same as those facing investigators who generate whole-cell hybrids; that is, only a few human chromosomes can be readily selected for and retained by use of selective media. A way to circumvent this problem is to insert a selectable marker into each individual human chromosome. In a previous paper (29) we described the stable integration of pSV2 gpt (19), a plasmid containing the E. coli sequence for xanthine-guanine phosphoribosyl transferase (XGPRT), into human chromosomes by the DNA-mediated gene transfer technique of Graham and van der Eb (9). Expression of XGPRT in HPRT-deficient human cells confers resistance to aminopterin in the hypoxanthine-aminopterin-thymidine (HAT) selection system of Littlefield (15) and therefore selects for retention of any chromosome containing the integrated plasmid sequences. In this paper we describe the isolation of human cell clones transfected with pSV2 gpt, each containing a single integrated copy of the plasmid, and transfer of single human chromosomes containing the integrated XGPRT marker from these clones, via microcell fusion into mouse recipient cells. This system is the basis for the construction of a library of interspecific microcell hybrid clones, each containing a single, biochemically selectable human chromosome.
Mapping of human genes has been facilitated by the development of human-rodent somatic cell hybrids in which various combinations of human chromosomes are retained within a background of rodent chromosomes (33). By constructing a panel of hybrid clones in which each clone contains a unique combination of human chromosomes, a gene coding for a specific product can be assigned to a particular chromosome by its presence or absence in the clone panel (4). Numerous human genes have been assigned to specific chromosomes with the use of these interspecific clone panels (22, 27), but an important extension of this system is the development of a complete set of hybrid clones, each containing a unique single human chromosome. Such a library of hybrids could be used to confirm gene assignments derived from the hybrid clone panels, investigate possible trans-acting or complementation effects from combinations of different human chromosomes found in the clone panels, and study gene dosage effects directed by one or more copies of a particular human chromosome. Hybrids containing single human chromosomes have been described previously (3, 13, 14) but generally speaking have represented fortuitous retention of the given chromosome rather than selective retention, and therefore, the human chromosome is eventually segregated from the hybrid. A few human chromosomes can be maintained in hybrids by virtue of naturally occurring selectable markers, for example, hypoxanthine phosphoribosyl transferase (HPRT) on the X chromosome, thymidine kinase on chromosome 17, and adenine phosphoribosyl transferase on chromosome 16. In the Chinese hamster ovary cell system, purine-requiring mutants which can be complemented by genes on human chromosomes 14 and 21 also have been isolated (12, 13). Unfortunately, there are very few other selectable markers available for the remaining chromosomes, and isolation of auxotrophic mutants which can be complemented by pro*
MATERIALS AND METHODS Cell lines and plasmids. D98/AH-2, an HPRT-deficient derivative of HeLa cells, was originally described by Szybalski et al. (30). A9, an HPRT-deficient mouse line derived from mouse L cells, was described by Littlefield (16). HT1080-6TG, an HPRT-deficient human fibrosarcoma
Corresponding author. 140
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SELECTIVE TRANSFER OF CHROMOSOMES TO CELLS
(5), was mutagenized to 6-thioguanine resistance from the HT1080 cell line described by Rasheed et al. (21). Microcell hybrids and rodent cells were maintained on Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (FCS) and 100 U of penicillin per ml. Parental human D98/AH-2 and HT1080 cells were maintained on Eagle minimal essential medium (MEM) supplemented with 5% calf serum plus 5% FCS and 100 U of penicillin per ml. Cells were tested routinely for mycoplasma contamination by culture methods and DAPI assay (23) and were found to be negative. The plasmid vector, pSV2 gpt, containing the E. coli gene encoding XGPRT was described by Mulligan and Berg (19). Transfection of human cells. The transfection of D98/AH-2 and HT1080-6TG cells was described in a previous paper (29) and is a modification of the calcium phosphate precipitation procedure of Graham and van der Eb (9). The site of integration of pSV2 gpt was determined by in situ hybridization (10). Individual colonies were selected from different dishes to ensure that each clone was independently derived. Blot hybridization analysis of transformants and microcell hybrids. High-molecular-weight genomic DNAs were digested with individual restriction enzymes. Digestions were performed at 37°C overnight in Rl buffer (100 mM Tris-hydrochloride [pH 7.5], 50 mM NaCl, 5 mM MgCl2, 100 ,ug of bovine serum albumin per ml) for EcoRI and in Hin buffer (10 mM Tris-hydrochloride [pH 7.5], 50 mM NaCl, 5 mM MgCl2) for all other restriction endonucleases. The digestions were terminated by incubation at 65°C for 10 min. The DNA digests were chilled, mixed with 5% glycerol-0.5 ,ug of bromophenol blue per ml, and loaded onto 0.8% agarose gels. Electrophoresis was carried out in TAE buffer (40 mM Tris-acetate [pH 7.9], 1 mM EDTA) at 0.25 mA/cm2 for 15 h. DNA fragments were then transferred onto nitrocellulose filters by the method of Southern (28). Filters were hybridized to heat-denatured 32P-labeled probe DNA (specific activity, 2 x 108 to 5 x 108 cpm/,ug of DNA) at 65°C overnight. Hybridization was performed in 5 x SET buffer (150 mM Tris-hydrochloride [pH 8.0], 5 mM EDTA, 750 mM NaCI) containing 0.02% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone, 0.5% sodium dodecyl sulfate, 10% dextran sulfate, and 100 ,ug of heat-denatured herring sperm DNA per ml as carrier. Filters were washed in 2x SET buffer (pH 8.0) containing 0.2% sodium dodecyl sulfate for 4 h at 65°C and in 3 mM Tris-base (pH 8.0) solution for 2 h at room temperature. Filters were dried and exposed to Kodak XAR-5 film with an intensifying screen at -70°C for a period of 2 to 8 days. Microcell-mediated chromosome transfer. Cells used as microcell donors were seeded into six 25-cm2 tissue culture flasks (Nunc) and incubated until 80 to 90% confluent. Colcemid (Calbiochem-Behring) was added to a final concentration of 0.2 ,ug/ml for D98 gpt transformants and 0.01 ,ug/ml for HT1080 gpt transformants. After 48 h colcemid was removed, and treated cells were allowed to recover in the presence of normal growth medium (MEM plus 10% FCS) for 2 to 4 h. For enucleation, flasks were filled to the neck with MEM containing 10 jig of cytochalasin-B (Sigma Chemical Co.) per ml. The flasks were preincubated at 37°C for 30 min, then placed in a JA-14 fixed-angle rotor containing 100 ml of water in each well for a cushion, and spun at 25,000 x g for 65 min at 34°C. Microcell pellets were removed from the flasks, pooled, and filtered through polycarbonate membrane filters (Nucle-
141
pore Corp.) in series, using 5.0- and 3.0-,um pore sizes, respectively. Typical yields of 1 x 107 to 2 x 107 microcells that were 3.0 ,um in diameter or smaller were routinely obtained. Microcells were fused to 90% confluent monolayers of mouse A9 cells by resuspending the microcell pellet in 2 ml of MEM plus 50 jig of phytohemagglutinin-P (Difco Laboratories) and then adding the suspension to recipient cells. Microcells were allowed to attach at room temperature for 20 min and then fused to recipient cells with 2 ml of polyethylene glycol (PEG-1450, 50% in MEM) for 60 s. The cells were quickly rinsed three times with phosphate-buffered saline to remove excess polyethylene glycol and then incubated for 12 h on Dulbecco modified Eagle medium plus 10% FCS. Fused cells were trypsinized and split into five 100-mm dishes on Dulbecco modified Eagle medium plus 10% FCS for 2 to 3 days. Selection medium, Dulbecco modified Eagle medium plus HAT (15), was added, and surviving clones were picked after 2 to 3 weeks, expanded, and analyzed for enzyme activity. Individual clones were picked from separate plates and, therefore, were considered to be derived from independent fusion events. HPRT enzyme assay. Enzyme activity was determined by agarose gel electrophoresis and is a modification of the method of Chasin and Urlaub (2). Briefly, cells to be assayed were trypsinized from a confluent 100-mm petri dish and lysed by three cycles of freeze-thawing. Supernatants of the cell extracts were run on a 1% agarose gel and incubated with [14C]guanine plus 5-phosphoribosyl 1-pyrophosphate for 90 min at 37°C. The labeled product ([14C]guanosine monophosphate) was precipitated into the gel with La(NO3)3, the excess labeled substrate was washed off, and the gel was exposed to X-ray film by PPO (2,5-diphenyloxazole) fluorography. Chromosome analysis. Prometaphase chromosome spreads were prepared by the method of Nichols(20). These spreads were analyzed by three different methods. First, chromosome identification was accomplished by G-banding, by the trypsin-Giemsa technique previously described by Worton and Duff (34). Banded spreads were photographed and destained for 15 min in methanol-acetic acid (3:1). The detained spreads were restained by the alkaline-Giemsa technique (G-11) of Friend et al. (8) to identify the single human chromosome in the rodent background. This method stains rodent chromosomes magenta, whereas human chromosomes stain pale blue. Preliminary chromosome identification derived from G-banding analysis was confirmed by human marker isoenzyme analysis by established electrophoretic procedures (11). Finally, prometaphase spreads were hybridized with either a tritiated human genomic DNA probe or tritiated pSV2 gpt DNA for in situ hybridization studies (10). TABLE 1. Transfection of HGPRT-deficient D98/AH-2 cells with
pSV2 gpta Avg no. of HAT-
Carrier DNA (20
Amt of pSV2 gpt (ng per
>g) D98/AH-2 D98/AH-2 D98/AH-2 Herring sperm Calf thymus Mouse A9
dish)
resistant colonies per dish
0 200 500 500 500 500
0 0.33 2.5 2.2 2.0 1.8
Cri
Transformants per pLg of DNA
1.6 5.0
4.4 4.0 3.6
a D98/AH-2 cells were transfected with purified pSV2 gpt plasmid DNA in the presence of carrier DNA by the calcium phosphate precipitation technique (details in the text). Colonies were scored after 21 days.
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SAXON ET AL.
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FIG. 1. Nature of integrated state of pSV2 gpt in D98 gpt and HT1080 gpt transformants. The transformant DNAs were digested with SstI, an enzyme that does not cut within the plasmid. Southern blot analysis with 32P-pSV2 gpt as probe showed that D98 gpt transformant 5 contains multiple integrants, whereas HT1080 gpt transformants 1 and 4 and D98 transformant 7 contain single integrants of pSV2 gpt.
RESULTS Transfection of human cells with pSV2 gpt results in stable integration and expression of plasmid DNA. D98/AH-2 and
HT1080-6TG (both HPRT-deficient) cells were efficiently transfected with E. coli gpt plasmid DNA (pSV2 gpt). An example of the transfection of D98/AH-2 is given in Table 1. Transfection efficiency was influenced by the concentration of plasmid DNA; the highest efficiency was obtained when 500 ng of plasmid DNA with 20 ,ug of homologous carrier DNA was used. Transformants selected in HAT medium were grown for several generations in nonselective (HT) medium and then placed back onto selective medium to score for stable transformants. About 20% of the original transformants were found to be unstable by the selection protocol described above (data not shown). Stable transformants were characterized by digestion of genomic DNA with the restriction endonuclease enzyme SstI to assess the number of integrated pSV2 gpt copies (Fig. 1). There are no SstI restriction sites within the plasmid, and therefore this enzyme cleaves only cellular DNA sequences outside of the integrated plasmid sequence. For single integrant clones 1, 4, and 7, a single band is seen on Southern blots when SstI-digested DNA is hybridized with 32P-pSV2 gpt. Clone 5 contains three integrated copies of plasmid DNA. Additionally, in situ hybridization of 3H-pSV2 gpt to metaphase spreads of chromosomes from either transfected cells or microcell hybrids localizes the site of integration of the plasmid sequence to a single site on one human chromosome (Fig. 2). The expression of Eco gpt in the transformants was examined by HPRT enzyme assays. The vast majority of the D98 gpt and HT1080 gpt transformants expressed Eco gpt (Fig. 3). The bacterial XGPRT activity can be readily distinguished from human HPRT activity by their different migration rates on the assay gel. Microcell-mediated chromosome transfer. Three transformants (HT1080 gpt transformant clones 1 and 4 and D98 gpt transformant clone 7) shown to contain a single copy of pSV2 gpt plasmid DNA were used as microcell donors.
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SELECTIVE TRANSFER OF CHROMOSOMES TO CELLS
Microcells were isolated (see above) and fused to HPRT-deficient mouse A9 cells. Microcell hybrids selected in HAT medium arose ca. 3 to 4 weeks after fusion, at a frequency of 1 x 10-6 to 2 x 10-6. The hybrids were picked with glass cloning rings, expanded, and assayed for expression of XGPRT activity. Of the hybrid clones, 75% were found to express the bacterial XGPRT activity, whereas 25% expressed no detectable activity of either form (bacterial XGPRT or mouse HPRT). One clone was found to express the wild-type mouse HPRT enzyme activity. The appearance of the clones which showed no detectable expression of either XGPRT or HPRT may be due to levels of expression which are too low to detect with this assay system. Alternatively, it is possible that these clones have become methotrexate resistant due to mechanisms other than expression of salvage-pathway HPRT enzymes. The single clone which was found to express the mouse form of HPRT was a rare revertant to wild-type activity, as has been seen by others (26, 32). Southern blot analysis was used to confirm that no rearrangements of the pSV2 gpt sequences had taken place after microcell fusion. Identical bands were seen (Fig. 4) for transformant versus the microcell hybrids when both were digested with EcoRI and HindIII, both of which cut once within the plasmid, and then probed with labeled pSV2 gpt. Chromosome analysis. Three microcell hybrids which expressed only bacterial XGPRT enzyme activity were analyzed for the presence of human chromosomes in the mouse A9 background by alkaline Giemsa (G-11) stain. The human chromosomes stain blue, whereas mouse chromosomes stain magenta. The lighter-staining chromosomes are human (arrowed) (Fig. 5). In a few microcell hybrids, more than one human chromosome could be seen in the mouse cells, but upon prolonged selection in HAT medium the extra human chromosomes were segregated out, whereas only the pSV2 gpt marked chromosome was retained. Trypsin-Giemsa banding was used to initially identify the human chromosomes retained in these three hybrids. Preliminary identification of chromosomes 4 and 22 was confirmed by isoenzyme marker analysis: phosphoglucomutase-2 for chromosome 4 and mitochondrial aconitase-1 for chromosome 22 (M. Smith, personal communication; data not shown). Chroa
b
c
d
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FIG. 3. Expression of bacterial XGPRT activity in transfected HT1080 cells. Extracts prepared from the transformants were electrophoresed in 1.0%o agarose gels, and the enzyme activity was detected by the incorporation of ['4C]guanine. Human HPRT activity from Hela cells (lanes a and g) and bacterial XGPRT activity from E. coli containing pSV2 gpt (lane e) were included as controls. Lane f contains D98/AH-2 cell extract as a negative control. Lanes b, c, and d show XGPRT activity from transformants 1, 4, and 7,
respectively.
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mosome 2 was confirmed by hybridization to the 1.0kilobase EcoRI-BamHI fragment of pNb-1, a plasmid containing N-myc related sequences previously localized to chromosome 2 (25) (data not shown). As seen in Table 2 the three series of microcell hybrids each contain a different human chromosome; the chromosome originating from D98 gpt clone 7 is autosome 2 and autosomes 4 and 22 from HT1080 gpt clones 4 and 1, respectively. The fact that the same chromosome is seen in each independent microcell hybrid clone derived from a given transformant underscores the stability of the integrated state of the pSV2 gpt plasmid. We have also, to date, not observed any fragmentation of the transferred human chromosomes. In situ hybridization. In situ hybridization was used to confirm that the pale-staining chromosomes were indeed of human origin. 3H-labeled total human genomic DNA was hybridized to metaphase chromosome spreads under stringent conditions. The exposed silver grains were seen predominately over the single human chromosomes (Fig. 6). No obvious areas of multiple grain clusters were noted over any of the mouse chromosomes, nor were subchromosomal fragments with associated grain clusters observed. Thus, in the cells analyzed it would seem that the only significant quantity of human DNA retained was found in the single intact chromosome.
DISCUSSION The purpose of the studies outlined here was to develop the means for generating a human chromosome library
144
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consisting of a series of mouse-human somatic cell hybrid clones, each containing a single specific human chromosome. We chose the microcell-mediated chromosome transfer technique (7) to transfer single human chromosomes from donor cells to recipient mouse cells. This method of chromosome transfer avoids the necessity of waiting for nonselectable human chromosomes to be segregated from interspecific whole-cell hybrids, while retaining only chromosomes containing a biochemically selectable marker. This method also avoids fragmentation of the transferred chromosomes seen in isolated metaphase chromosome transfer techniques (17). The problem of selection of microcell hybrids containing single human chromosomes has been overcome by transfecting human cells with pSV2 gpt. There are several properties of these transformant cells that makes them particularly useful for these studies. First, single copies of pSV2 gpt
have randomly integrated into different chromoSo far, we have not observed any preferential integration into a particular chromosome in the various transformants we have examined. This suggests that there should be no problem in obtaining clones that carry an integrated plasmid in each human autosome and sex chromosome, providing that we isolate a large enough sampling of transformants. Also, the integration of the plasmid appears to be stable, with no rearrangments or loss of plasmid during prolonged culture in selective medium. The expression of the plasmid also is stable, with no gradual or sudden loss of XGPRT activity. This stable expression allows for selection of the microcell hybrid in HAT medium and for selective retention of the single human chromosome containing the integrated pSV2 gpt. Another useful feature of this system is that the chromosome containing the integrated pSV2 gpt plasmid can be
appear to somes.
SELECTIVE TRANSFER OF CHROMOSOMES TO CELLS
VOL. S, 1985
145
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selected against by placing the hybrid cells in culture medium containing 6-thioguanine. Thus, for any given individual human chromosome this procedure provides a selective system for the retention and loss of the chromosome in a rodent background. It should be noted that, whereas we used the HPRT-deficient A9 cells as the fusion partner, dominant selection also can be used as described by Mulligan and Berg (19). Wild-type mammalian cells will not grow in the presence of HAT with added mycophenolic acid. The bacterial XGPRT activity circumvents this block when the above medium is supplernented with xanthine. This system should aid human gene mapping studies by providing panels of microcell hybrids containing single selectable human chromosomes, which represent the entire human karyotype. These hybrids can be used as a source of single human chromosome DNA for creating lambda or TABLE 2. Identification of Human Chromosomes in Microcell
Hybrids Microcell Trnso n R n Transformant hybrid Recipient hyrd
D98 gpt clone 7
A9
7/A9.5 7/A9.6 71A9.14
Human chromosome comse 2 (18,20)a
2 2
HT1080 gpt clone 4
A9
JS4/A9.1 JS4/A9.2 JS4/A9.3
4 4 4
HT1080 gpt clone 1
A9
JS1/A9.4 JS1/A9.5 JSI/A9.6
22 22 22
Indicates a clone which initially contained more than one human chromosome but, upon further passages under HAT selection conditions, lost all human chromosomes except the one containing the integrated pSV2 gpt sequence.
cosmid libraries of single human chromosomes. Additionally, these hybrids can be used to create a series of subchromosomal fragments of specific human chromosomes by treating a particular hybrid with chromosome-fragmenting chemicals or radiation. Finally, these hybrids can be used to identify chromosome-specific sequences such as discrete single-copy genes or probes for restriction fragment length polymorphisms (1). Although these experiments have been carried out with the aneuploid D98/AH-2 and pseudo-diploid HT1080 transformed cell lines as chromosome donors, there is no reason to suspect that the same approach cannot be used with normal diploid fibroblasts as donors. We have initiated these studies by using the protoplast fusion procedure of SandriGoldin et al. (24) to transfect human fibroblasts with pSV2 gpt. It is also possible to use selective markers other than Eco gpt for dominant selection. We have successfully transfected HT1080 cells with pSV2 neo, a plasmid which encodes resistance to the neomycin analog G418, and subsequently used these transformants as microcell donors of single human chromosomes (unpublished data). Sequential microcell fusions with transformants containing different dominant selectable markers should make it possible to examine chromosome (gene) dosage effects and the interactions between two or more different chromosomes with respect to regulation of gene expression. While these studies were in progress a similar study was reported (31) in which human chromosome 17 containing integrated copies of pSV3 gpt, a transforming plasmid, was transferred to a mouse teratocarcinoma cell line by microcell fusion. ACKNOWLEDGMENTS We thank Moyra Smith for her help in identifying the human chromosomes and for performing the isoenzyme marker analysis.
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This work was supported by Research Grants from the National Cancer Institute (CA 19401 and CA 34114) and a grant from the Council for Tobacco Research-USA (no. 1475). E.J.S. is a recipient of Research Career Development Award CA00271 from the National Institutes of Health.
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