| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Use of a human minichromosome as a cloning and expression vector for mammalian cells
Introduction
Results
Characterization of human minichromosome MC1
Targeting the human IL-2 cDNA to the pericentromeric region of MC1
CHO-MC1 transfected clones express the IL-2 gene
Presence of the IL-2 gene marker and stability of MC1 in the ILS clones
Generation of human-mouse radiation hybrids containing MC1-IL2
IL-2 expressed in the T-IL hybrids protects CTLL cells from apoptosis
Stability of MC1-IL2 in the T-IL hybrids
Discussion
Materials And Methods
Plasmids
Cell cultures and DNA transfection
PCR amplification
Preparation of high molecular weight genomic DNA and CHEF analysis
Fluorescence in situ hybridization and detection
Immunofluorescence staining
Cell irradiation and fusion
Acknowledgements
References
Use of a human minichromosome as a cloning and expression vector for mammalian cells
Received January 6, 1999; Revised and Accepted April 30, 1999
A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1-IL2). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.
INTRODUCTION
In recent years efforts have been underway to develop mammalian artificial chromosomes (MACs) for use both in basic and applied biology. MACs can be useful tools for the understanding of chromosome structure and function and will provide a means for introducing fragments of DNA large enough to drive full expression of proteins into mammalian cells and organisms, with considerably more control and fewer risks than with the currently utilized integrating vectors. The development of MACs is expected to lead to useful biotechnological applications in animal husbandry and agriculture and to effective gene therapy (1-5).
MACs can be circular, and therefore lack telomeres, or linear, with free telomeric ends. A number of circular MACs based on viral vectors have been developed (6-10). Such vectors suffer from the limitations of requiring viral proteins for their replication. Circular molecules containing the centromeric region of the human Y chromosome were able to replicate in mouse cells, but function of the centromeric sequences was impaired (11). More attention has been devoted recently to the development of linear MACs, following a bottom-up approach in which the elements necessary for MAC function are prepared and assembled (12,13). An alternative approach (top-down) is to reduce an existing chromosome to a minimal size by X-ray irradiation or telomere fragmentation (14-16). Given the small size of these molecules, as opposed to that of natural chromosomes, MACs can also be called minichromosomes. An attempt to construct a virus-based linear MAC following the bottom-up approach was unsuccessful, presumably because the replication origin of the circular virus SV40 did not function in a linear molecule (17). Two recent studies have shown that human centromeric sequences and telomeric repeats can assemble to form MACs, some of which can segregate accurately (12,13). The feasibility of using chromosomes of reduced size as MACs has been suggested (2,3,18), but their ability to be used as cloning or expression vectors has not been tested. It has been shown, however, that human chromosomes or large chromosome fragments can be introduced into mouse embryonic stem cells to produce chimeric mice, where the chromosomes are retained with high stability and the human genes are expressed in a proper tissue-specific manner (19). It is unlikely, however, that it will be possible to manipulate such large molecules in such a way that they will serve as useful cloning and expression vectors.
In the present study we report experiments aimed at the utilization of a small, and therefore manageable, minichromosome, designated MC1, as a cloning and expression vector for mammalian cells and as a possible vector for gene transfer. MC1 is a 5.5 Mb human minichromosome derived from human chromosome 1 and is present in a CHO hybrid cell line (20,21). The neo gene for selection of transfectants and the cDNA of the human interleukin 2 (IL-2) gene were introduced into MC1. The choice of the IL-2 gene for evaluating the efficacy of MC1 as an expression vector for mammalian cells was based on several considerations. IL-2, a lymphocyte growth and differentiation factor, is one of the most potent known antitumor cytokines (22-25). Both the injection of recombinant IL-2 and its release by cytokine gene-engineered cells result in the activation of a strong antitumor reaction. However, the systemic toxicity of injected IL-2 and its rapid degradation suggest that the implantation of genetically modified somatic cells represents the best way to achieve effective concentration of the cytokine. Two kinds of cells have been genetically modified for delivery of IL-2, tumor cells and T lymphocytes: the former for provoking tumor rejection and immune memory and the latter for enhancing immunoreactivity of tumor infiltrating lymphocytes (24,25). We show in this study that both these categories of cells can be engineered by means of a minichromosomal vector to express IL-2.
RESULTS
Characterization of human minichromosome MC1
MC1 (20) was furnished in the CHO hybrid cell line XEW8.2.3, where it is present as the only free human chromosome. MC1 was obtained after irradiation of a CHO-human hybrid containing chromosome 1 and has retained a small portion of the short arm and of the centromere representing ~1% of the size of the parent chromosome (21). It has also been shown that MC1 is completely stable in CHO cells, as it was shown to be present in all metaphase spreads, as evidenced by FISH analysis, both under selection and without selection, and at all times (21). In our hands, we have always found a value of 65-70% for the presence of MC1 in CHO metaphase spreads, under all conditions and over a period of ~10 years. We do not have an explanation for our inability to detect a significant fraction of the minichromosomes by FISH analysis. In order to determine the size of MC1, intact DNA from CHO cells containing either human chromosome 1 or MC1 was separated by CHEF electrophoresis and hybridized to a satellite 2 (Sat2) DNA probe as described in Materials and Methods (Fig. 1). Human chromosome 1 does not enter the gel, whereas the MC1 molecule penetrates the gel reaching a position very similar to that of the largest Schizosaccharomyces pombe chromosome (5.7 Mb) run on the same gel. Further proof of the linearity of the minichromosome was provided by experiments that showed that the electrophoretic mobility of the molecule was not modified after DNA digestion with DNase under conditions that produce nicks (Fig. 1). Furthermore, digestion of CHO-MC1 DNA embedded in agarose plugs with either of the two rare-cutting enzymes BglII and NdeI and hybridization with total human DNA-, Sat2- and alphoid 1-specific DNA probes yielded large molecular weight DNA fragments that add up to 5.5 Mb (data not shown). These results demonstrate that the molecule is linear and confirm the size determined by CHEF analysis.
Figure 1. CHEF analysis of the minichromosome MC1. Intact DNA was prepared in agarose plugs from the indicated cell lines. GM13139 is a CHO line containing the entire human chromosome 1 and ILS is the ILS22 clone that contains the modified version of MC1, MC1-IL2. After separation, the DNA was transferred to a Hybond membrane and hybridized to a 32P-labeled Sat2 probe. The amount of DNase used to digest the ILS22 samples is indicated in U/ml. The first lane on the left contains S.pombe chromosome DNA markers.
Targeting the human IL-2 cDNA to the pericentromeric region of MC1
It was shown that the neo gene can be targeted to the centromeric region of a human minichromosome derived from chromosome 9 by co-transfection with chromosome 9-specific satellite DNA, and that the neo gene, inserted in the centromeric region of the minichromosome, can be expressed in spite of its being embedded in centromeric heterochromatin (18). We have used an analogous approach to introduce both the neo and the IL-2 genes into the MC1 minichromosome (Materials and Methods). In view of the fact that Sat2 pericentromeric sequences are considerably more abundant in MC1 than alphoid DNA, we targeted the marker genes to the Sat2 DNA, by co-transfecting the plasmids pUC1.77neo (26,27) and pBC12/CMV/IL-2 (28) into CHO-MC1 hybrid cells. It has been observed in mammalian cells that unrelated DNA molecules end join efficiently in vivo via non-homologous recombination (29,30); thus, plasmids pUC1.77neo and pBC12/CMV/IL-2 were co-transfected in linear form (Materials and Methods) to increase the probability that both the neo and IL-2 genes would integrate into the pericentromeric region of MC1.
CHO-MC1 transfected clones express the IL-2 gene
A large number of G418-resistant CHO-MC1 clones were obtained after co-transfection and 25 of them, designated ILS clones, were isolated and assayed with the CTLL stimulation of proliferation assay to estimate the amount of IL-2 secreted into the culture medium. About 3 × 105 cells of each ILS clone were plated in 35 mm dishes and after 48 h growth the medium was collected, centrifuged to remove cellular debris and diluted 1:1 with fresh RPMI (conditioned medium). Twenty-five CTLL cultures were set up in conditioned medium at a concentration of ~3 × 104 cells/ml, without addition of exogenous IL-2. As a control, two cultures were set up with exogenous IL-2 added at concentrations of 5 and 10 U/ml. After 48 h, cell growth and viability were measured by counting viable cells with a hemocytometer. Four of the 25 CTLL cultures had reached a density of ~1 × 105 cells/ml, slightly lower than that of the control culture containing 5 U/ml IL-2. Taking into account the 1:1 dilution of the growth medium, it was estimated that the amount of IL-2 produced by the ILS clones was of the order of 8 U/ml. Four clones, ILS8, ILS11, ILS12 and ILS22, were chosen for further analysis. The remaining 21 ILS clones did not yield IL-2 levels sufficient for CTLL growth and were not analyzed further.
Presence of the IL-2 gene marker and stability of MC1 in the ILS clones
PCR amplification of the four ILS DNAs and of control CHO-MC1 DNA, using human Alu primers, gave identical amplification patterns consisting of multiple DNA bands ranging in size from 200 to ~2500 bp (data not shown), demonstrating the presence of MC1 in the four ILS clones. PCR amplification of the same ILS DNA extracts using IL-2 primers generated in all cases a single 500 bp DNA fragment (Fig. 2A). IL-2 RT-PCR of total RNA and of mRNA extracted from ILS22 cells also yielded a 500 bp DNA band that hybridized with an IL-2 probe (Fig. 2B). The RNA-specific origin of the RT-PCR products was demonstrated by RNase treatment of the mRNA sample. Similar experiments performed with mRNA from the other ILS clones gave the same results (Fig. 2C), demonstrating that the 500 bp fragment was indeed the IL-2 transcript.
Figure 2. PCR and RT-PCR amplification of the IL-2 gene in the ILS clones. (A) Ethidium bromide stained agarose gel of the IL-2 PCR reactions carried out with DNA samples from the indicated clones. pBC12/CMV/IL-2 is the IL-2-containing plasmid. The asterisk indicates the IL-2 amplified DNA fragment. (B and C) Hybridization of the RT-PCR products with an IL-2 probe. The reactions in (B) have been performed with ILS22 RNAs and those in (C) with mRNA samples from the indicated clones.
A double colored fluorescence in situ hybridization was performed on ILS22 metaphase spreads using IL-2 and Sat2 DNA as probes. Detection of these probes revealed the co-localization of both DNAs in the smallest DNA element present in the metaphases (Fig. 3A), thus proving that this molecule was indeed MC1. Furthermore, FISH analysis of ILS22 chromosomes with either the Sat2 or IL-2 probes revealed only one fluorescent signal that localized to the minichromosome, as shown in the two examples given in Figure 3B. A similar result has been obtained with a neo probe (data not shown). The modified MC1 contained in ILS22 was selected for further study and designated MC1-IL2.
Figure 3. Analysis of MC1-IL2 using fluorescence in situ hybridization. (A) Dual color FISH of metaphase chromosomes from ILS22 cells with digoxigenin-labeled Sat2 DNA and biotin-labeled IL-2 DNA as probes. (a) Chromosomes counterstained with DAPI; the arrow indicates the minichromosome; (b) Sat2 probe revealed with anti-DIG-FITC; (c) IL-2 probe revealed with avidin-Cy3. Fluorescent signals were pseudocolored with a CCD camera and a digital imaging system. (B) Metaphase chromosomes from ILS22 cells hybridized in situ to biotin-labeled Sat2 DNA (a) and biotin-labeled IL-2 DNA (b). Chromosomes were counterstained with propidium iodide and the probes were revealed with avidin-FITC. Arrows indicate the minichromosome. (C) An ILS22 chromosome plate stained with DAPI (a); treated with CREST (b); hybridized with a digoxigenin-labeled Sat2 DNA (c).
The fraction of ILS22 cells containing MC1-IL2 was determined in metaphase spreads stained with DAPI. This analysis was carried out 50 and 200 mean population doublings (MPD) after transformation in the absence of G418 selection, screening ~100 metaphases per time point. In both cases the minichromosome was retained in 70% of the cells, a value consistent with the frequency of MC1 in CHO cells, as determined by us. More than one copy of MC1-IL2 was never observed. The same result was obtained by scoring for Sat2-hybridized MC1-IL2 in ILS22 metaphase spreads after 200 MPD. These results strongly indicate that MC1-IL2 has an active centromere and this activity is confirmed by centromere antigen staining with CREST autoimmune sera (Fig. 3C).
Generation of human-mouse radiation hybrids containing MC1-IL2
Having established that MC1-IL2 can function as a vector for IL-2 expression, we transferred the minichromosome into an IL-2-dependent cell line in order to test its ability to express IL-2 in amounts sufficient to support cell growth.
Cell fusion experiments were performed between irradiated ILS22 and the recipient murine IL-2-dependent CTLL cells. The optimal radiation dose was determined on the basis of the data of Sidén et al. (31) and the function developed by Goss and Harris (32) relating the probability of breakage between two loci to the X-ray dose. In order to kill the cells with little likelihood of causing damage to MC1-IL2, the ILS22 donor cells were exposed to a radiation dose that would produce chromosomal fragments not much smaller than 5 Mb, the size of MC1. Radiation hybrids (Materials and Methods) were selected for the capacity to grow in RPMI medium without exogenous IL-2 addition.
Among the 142 hybrid CTLL clones obtained, 120 (84%) grew in RPMI for at least 17 MPD, reaching a density of 1 × 105 cells/ml. After this time period, the growth rate decreased and eventually the cells ceased to grow. Three clones, T-ILa, T-ILb and T-ILc, were selected for further experiments and were routinely propagated in RPMI medium containing a low (3-5 U/ml) concentration of IL-2.
To verify the presence of markers in the radiation hybrids, DNA extracted from the three T-IL hybrids after 20, 50 and 80 MPD was PCR amplified for Alu and IL-2 sequences. As a control, DNA extracted from the donor and the recipient cells at the same time points was also analyzed. As expected, the Alu-PCR amplification pattern characteristic of MC1 was obtained with all DNAs with the exception of CTLL DNA and the amplified DNA fragments hybridized with the Alu probe (Fig. 4A and B). IL-2 PCR amplified the same 500 bp DNA fragment from T-IL and ILS22 DNA, confirming the presence of the IL-2 gene (Fig. 5A). These results strongly suggest that the minichromosome structure was not altered by irradiation, although it is not possible to rule out the hypothesis that small rearrangements may have taken place. Moreover, RT-PCR on total RNA extracted after ~20 and 50 MPD from the same clones using IL-2 primers demonstrated that the IL-2 gene was transcribed and hybridization of the dried gel with an IL-2 probe confirmed the identity of the IL-2 RT-PCR product (Fig. 5B and C). mRNA was extracted from the T-ILc clone and IL-2 RT-PCR was carried out with samples treated and not treated with RNase. The absence of the RT-PCR product in the RNase-treated sample demonstrated the RNA-specific origin of the 500 bp DNA band (data not shown). The presence of MC1-IL2 in T-ILc cells was demonstrated by FISH using a Sat2 DNA probe. After 50 MPD, MC1-IL2 was detected in ~30% of the metaphases analyzed, while after 80 MPD metaphases containing MC1-IL2 were very rare. Alu and IL-2 PCR-amplified products were obtained with DNA extracted at 20 and 50 MPD, whereas almost no amplification was obtained with DNA extracted at 80 MPD (data not shown). By increasing the amount of the latter template DNA 10-fold, amplification products were obtained, indicating that DNA extracted at 80 MDP contains a small amount of IL-2 template. These results suggested that in the T-IL cultures MC1-IL2 is being progressively lost from the T-IL cells.
Figure 4. PCR amplification products of the Alu repetitive sequences in the T-IL clones at 50 MPD. (A) Ethidium bromide stained agarose gel. MW, HindIII-digested [lambda] DNA; lane 1, ILS22; lane 2, CTLL; lanes 3-5, T-ILa, T-ILb and T-ILc. In lane 2 a DNA band that is a non-specific CTLL DNA amplification product is visible. (B) Hybridization of the gel shown in (A) with an Alu probe.
Figure 5. PCR and RT-PCR amplification of the IL-2 gene in the T-IL clones at 20 MPD. (A) Ethidium bromide stained agarose gel of the PCR amplification products of DNA extracts with the IL-2 primers. Lane 1, HindIII-digested [lambda] DNA; lane 2, plasmid pBC12/CMV/IL-2; lane 3, ILS22; lane 4, CTLL; lanes 5-7, T-ILa, T-ILb and T-ILc. (B) Ethidium bromide stained agarose gel of the RT-PCR amplification products obtained with IL-2 primers of RNA extracts. MW, HindIII-digested [lambda] DNA; lane 1, ILS22; lane 2, CTLL; lanes 3-5, T-ILa, T-ILb and T-ILc. (C) Hybridization of the gel shown in (B) with an IL-2 probe.
IL-2 expressed in the T-IL hybrids protects CTLL cells from apoptosis
Considering that withdrawal of IL-2 from the CTLL culture medium induces apoptosis (33-35), an experiment was performed to verify whether the IL-2 produced by MC1-IL2 in the T-IL hybrids could protect the cells from this phenomenon, indicating a normal physiological role for the cytokine produced by the cells. Aliquots of ~2 × 106 CTLL and T-ILc cells grown for 50 MPD in the presence of IL-2 were washed twice in serum-free medium, plated in RPMI medium without IL-2 and DNA was extracted at regular time intervals. DNA analysis on agarose gels (Fig. 6) revealed that the characteristic ladder pattern of oligonucleosomal fragments was present in the CTLL DNA 7 h after IL-2 withdrawal. The appearance of the apoptotic DNA pattern was delayed in T-ILc for 24-30 h, demonstrating that the endogenous production of IL-2 was sufficient to postpone activation of the cell suicide program.
Figure 6. DNA fragmentation kinetics of IL-2-deprived CTLL cells and of the T-ILc hybrid. Ethidium bromide stained agarose gel of DNA purified from CTLL and T-ILc cells grown for the times indicated (0, 7, 24, 30 and 50 h). The molecular weight marker on the left is HindIII-digested [lambda] DNA and on the right the 100 bp DNA ladder (Gibco BRL).
Stability of MC1-IL2 in the T-IL hybrids
In order to confirm loss of MC1-IL2 in T-ILc cells, we determined the cloning efficiency of such cells after 5, 50 and 80 MPD of growth in the presence of IL-2. T-ILc cells were subcloned from the culture at the concentration of 0.5 cells/well into Terasaki plates without IL-2. After 2 weeks incubation the number of clones that had grown up was determined and the value corrected for cloning efficiency. This experiment showed that after 5 and 50 MPD, 73 and 26% of the cells produced viable clones. No viable clones were obtained from cells at 80 MPD. Lack of cell growth could be due to loss of MC1-IL2 or loss of IL-2 function with increasing MPD. To distinguish between these two possibilities, the amount of MC1-IL2 DNA present at 80 MPD in T-ILc cells was measured by DNA hybridization. DNA extracts of ILS22 and T-ILc at 80 MPD were run on an agarose gel that was dried and hybridized with a total human DNA probe. The hybridization intensity measured with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and compared with known amounts of ILS22 DNA showed that the MC1-IL2 DNA in the T-ILc extract amounted to only 2% of the DNA contained in ILS22, consistent with the notion that MC1-IL2 is progressively lost. The three values obtained for MC1-IL2-containing cells after 50 MPD led to an average per generation segregating efficiency of 97%, as calculated according to Murray and Szostak (36).
DISCUSSION
The present study was aimed at determining whether a 5.5 Mb minichromosome, MC1, can be used as a cloning and expression vector for mammalian cells and possibly organisms. Other workers have shown that genes for selection in mammalian cells can be cloned into the centromere of minichromosomes and that such genes can express their functions in spite of their being embedded in centromeric sequences (18,37). We have used a similar approach to insert both the neo and the IL-2 genes into the MC1 minichromosome, resulting in the production of a set of MC1-IL2 minichromosomes. Four out of 25 co-transfected clones that expressed the neo gene were also found to express the IL-2 gene. The level of expression of IL-2 was similar in all four clones. Whether this is due to the presence of similar gene copy numbers in the different clones, to the location of the gene in MC1 or to other functions cannot be established at present. The IL-2 produced by the transfectants was biologically active, since IL-2-dependent CTLL mouse cells grew when supplemented with supernatants of the transfectant cultures. The dual color fluorescence in situ hybridization analysis of MC1-IL2 showed that the IL-2 gene was present on MC1, attesting that co-transfection with Sat2 sequences results in correct targeting of the IL-2 gene. In order to test the feasibility of utilizing the minichromosome as a vector for genes that can complement cellular functions, including functions required for gene therapy, MC1-IL2 was introduced by cell fusion into CTLL cells. We have shown that MC1-IL2-dependent expression of the cytokine allowed the selection of human-CTLL hybrids (T-IL cells) that had acquired the minichromosome by culturing the fused cells in medium lacking IL-2. We were also able to demonstrate that IL-2 was produced at a level sufficient to stimulate growth of the hybrids for as many as 17 MPD and that IL-2 expression caused a significant delay in the onset of apoptosis in the cells.
We detected the presence of a minichromosome in CHO cells, containing either MC1 or MC1-IL2, in 70% of metaphase spreads from cells grown either under or without selection, indicating that the subset of chromosome 1 centromeric sequences present in the minichromosomes ensure mitotic stability, even when foreign genes have most likely been introduced into the pericentromeric region.
The MC1-IL2 minichromosome is somewhat less stable in CTLL cells, where it exhibits a mitotic instability of 3% per MPD in the absence of selection. It remains to be seen whether this instability is due to the response of the CTLL cells to errors in chromosome segregation or whether it reflects an incapacity of the MC1 centromere to function in a completely normal fashion in mouse cells, making this minichromosome sensitive to the cellular environment. Other workers have observed a similar phenomenon: a circular human minichromosome containing the entire Y centromere fails to segregate properly in mouse fibroblasts (11) and a minimal human Y chromosome of 4 Mb in size was found to be unstable in a mouse ES cell background (12).
The results reported in this paper show that the MC1 minichromosome system can be used as a cloning and expression vector and can be engineered for IL-2 delivery in two different types of cells, fibroblasts and lymphocytes, making molecules of this class promising candidates for vectors to be used for therapeutic purposes.
MATERIALS AND METHODS
Plasmids
Plasmid pUC1.77 (4.2 kb) contains a 1.77 kb human satellite III DNA fragment specific for chromosome 1 (26) that was recently redefined as satellite 2 DNA (27). Insertion of a 2.6 kb gel-purified DNA fragment containing the neo gene into the BamHI site of the polylinker of pUC1.77 gave rise to pUC1.77neo (6.8 kb). Plasmid pBC12/CMV/IL-2 (28) contains a 491 bp DNA fragment that extends from 1 bp 5[prime] of the AUG initiator codon to 34 bp 3[prime] of the TGA terminator codon of the human IL-2 cDNA, cloned under the CMV-IE promoter. Restriction endonucleases and DNA modifying enzymes (Promega, Madison, WI and New England Biolabs, Milano, Italy) were used according to the suppliers' specifications.
Cell cultures and DNA transfection
CHO-MC1 cells (kindly supplied by Immo E. Scheffler, University of California at San Diego, La Jolla, CA) were cultured in DMEM medium supplemented with 10% fetal calf serum, 10 mM essential amino acids, 2 mM L-glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin (all products were from Biological Industries, Haemek, Israel) at 37°C under a humidified atmosphere containing 5% CO2. The IL-2-dependent lymphoblastoid murine cell line CTLL was grown in RPMI-1640 medium (Gibco BRL, San Giuliano Milanese, Italy) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin and 10 U/ml IL-2 (Boehringer Mannheim, Monza, Italy). Cells were grown in 25 cm2 flasks at 37°C under a humidified atmosphere containing 5% CO2 to a density of 2 × 105 cells/ml and, at that concentration, immediately diluted 5-fold into fresh RPMI medium.
CHO-MC1 transfection was carried out by electroporation using a Bio-Rad (Roma, Italy) apparatus at 250 V, 960 mF. Exponentially growing cells were washed twice in electroporation buffer (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.8 mM NaH2PO4, 0.7 mM Na2HPO4, 1 g/l glucose) at 4°C and resuspended at a concentration of 3 × 106 cells/ml. One milliliter aliquots were mixed in a 0.4 cm pre-chilled cuvette with 3 mg of linearized pUC1.77neo, obtained by partial BamHI digestion, and 3 mg of PvuI-linearized pBC12/CMV/IL-2. The mixture was incubated on ice for 10 min and, following electroporation, the cells were diluted into fresh medium and seeded in 100 mm Petri dishes (Dasit S.p.a., Roma, Italy). After 24 h the selective agent G418 (Sigma, Milano, Italy) was added at a concentration of 1 mg/ml. A number of resistant clones that had appeared by 8 days after transfection were collected, named ILS clones and chosen for further analysis.
PCR amplification
The human IL-2 cDNA was amplified by PCR with the IL2 F (5[prime]-ATGTCAAGGATGCAACTCCTGTCTT-3[prime]) and IL2 R (5[prime]-GTCAGTTGTTGAGATGATGCTTTGAC-3[prime]) primers, 200 ng genomic DNA as template and 1.2 mM MgCl2 buffer. The amplification conditions were: 4 min at 95°C; 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min; 7 min at 72°C. The Alu primer 5[prime]-GCGGCCGCTTGCAGTGAGGCCTAGAT-3[prime] was used under the following conditions: 1.2 mM MgCl2 buffer; 1 cycle at 95°C for 4 min; 30 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 3 min; 1 cycle at 72°C for 7 min. Taq polymerase was from Perkin Elmer (Roma, Italy). Total RNA was extracted from mid log phase cells using the RNeasy mini kit (Quiagen, Firenze, Italy) and mRNA purifed with Oligotex spin columns. Aliquots of total RNA treated with DNase and mRNA, with and without RNase treatment, were used for RT-PCR of the IL-2 gene with Tth DNA polymerase (Boehringer Mannheim). Conventional procedures were used to label the probes and, when the Alu and IL-2 PCR amplification products were used in hybridization experiments, they were purified through a S-200 MicroSpin column (Pharmacia Biotech, Milano, Italy) prior to labeling.
Preparation of high molecular weight genomic DNA and CHEF analysis
CHO-MC1 and ILS22 cells grown to confluency in 100 mm plates were removed with trypsin/EDTA, washed twice with phosphate-buffered saline (PBS) and resuspended at a concentration of 5 × 107 cells/ml in L buffer (10 mM Tris-HCl pH 7.4, 100 mM EDTA, 20 mM NaCl). The suspension was then added to an equal volumne of 1% low melting point agarose (Boehringer Mannheim) and allowed to solidify at 4°C. After overnight incubation at 50°C in NDS buffer (10 mM Tris-HCl pH 7.5, 200 mM EDTA, 1% N-lauroyl sarcosine, 1 mg/ml proteinase K) the plugs were washed thoroughly in 50 mM EDTA and stored at 4°C. Digestion of genomic DNA in agarose plugs with BglII and NdeI (Boehringer Mannheim) was carried out overnight at 37°C using the supplier's specifications. Electrophoresis of intact DNA in agarose plugs was carried out for 336 h at 0.75 V/cm in 0.5 % agarose/0.5× TAE, pulse time 2 h, angle 120°, in a CHEF MAPPER apparatus (Bio-Rad). The gel was then stained with ethidium bromide, denatured, neutralized and transferred to a Hybond-N membrane (Amersham, Milano, Italy). Hybridization with the 32P-labeled Sat2 DNA probe was carried out at 65°C according to standard methods (39). DNase treatment of DNA plugs was performed in 10 mM MgCl2, 50 mM Tris-HCl pH 7.6, 0.1 mg/ml BSA at 25°C for 1 h (40).
ILS22 agarose plugs were also used to determine the copy number of the IL-2 gene in the minichromosome. Digestion and hybridization with an IL-2 probe strongly suggest that a single copy of the gene is present in MC1-IL2 (data not shown).
Fluorescence in situ hybridization and detection
Cultured cells grown to 60% confluence in 100 mm plates were incubated for 3 h in fresh medium containing 100 ng/ml colcemid (Gibco BRL) and then treated according to standard procedures with 0.04 M KCl, 0.025 M Na citrate hypotonic solution followed by fixing in methanol/acetic acid (3:1). T-ILc cells were grown in RPMI medium in 25 cm2 flasks and then treated according to the procedure described above. The IL-2, Sat2 and neo purified DNA fragments used for FISH were labeled using a random primed DNA labeling kit and Bio-16-dUTP. Metaphase spreads were treated according to the method described previously (41) and hybridization was detected using avidin conjugated to fluorescein isothiocyanate (FITC). The IL-2 DNA fragment used for the dual color in situ hybridization was labeled by PCR amplification using Bio-16-dUTP and detected with avidin conjugated to Cy3 (Amersham). Sat2 DNA was labeled with a random primed DNA labeling kit using digoxigenin-11-dUTP and detected with anti-digoxigenin conjugated with fluorescein. Staining with DAPI (100 ng/ml) was in PBS at room temperature. Images were acquired using a Zeiss Axioscop fluorescent photomicroscope and a CCD camera.
Immunofluorescence staining
Colcemid-treated ILS22 cells were washed in PBS and resuspended in 0.075 M KCl at a concentration of 5 × 104 cells/ml. Aliquots (500 ml) were cytocentrifuged at 755 g for 10 min. The slides were lysed in 120 mM KCl, 20 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100 at room temperature for 15 min, after which they were fixed in absolute methanol at -20°C for 30 min. Following two washes in PBS + 0.05% Tween-20 (PBST), the preparations were incubated for 30 min at room temperature in blocking solution (20% fetal bovine serum in PBST) and washed in PBST. Human autoimmune sera, diluted 1:1 in PBST, was added to the slides and after 2 h incubation at room temperature they were washed in PBST twice. The antibody signal was detected with a secondary antibody conjugated with the Cy3 fluorochrome. The chromosomes were counterstained with DAPI (100 ng/ml) and the slides were analyzed with a Zeiss Axioscop fluorescent photomicroscope. The chromosomes of the same slides were fixed with acetic acid/methanol (1:3) and hybridized with a digoxigenin-11-dUTP-labeled Sat2 probe. The hybridization signal was detected with anti-digoxigenin conjugated with fluorescein. After chromosome counterstaining with DAPI, the fluorochromes were detected using specific filters and the images acquired with a Kodak DC120 digital camera.
Cell irradiation and fusion
About 1 × 106 ILS22 cells containing the modified MC1 were washed three times in serum-free medium, resuspended in 0.5 ml ice-cold RPMI and exposed to 1000 rad/min for 10 min (Co 60-Cell 220 AECL). The donor cells were kept on ice during this treatment until fused with CTLL recipient cells. Fusion was carried out according to Sidén et al. (31) with minor modifications. Briefly, ~2 × 106 CTLL cells were mixed with the irradiated ILS22 cells and, after centrifugation at 200 g for 5 min, 200 ml of 50% polyethyleneglycol 1300-1600 (Sigma) and 50% RPMI were added to the pellet over a 1 min period, followed by addition of 5 ml RPMI containing 10 U/ml IL-2. The medium was added very slowly over a 5 min period. Cells were then seeded at various concentrations in 24-well plates and, 48 h later, were transferred to new plates in order to separate the CTLL cells growing in suspension from the ILS22 cells adhering to the wells. Eight days post-fusion, aliquots of the CTLL cells that had survived the treatment were frozen as polyclonal stocks and one aliquot was cloned in Terasaki plates. The post-fusion CTLL cells were diluted and aliquoted into the wells at a concentration of 0.5 cells/well, so as to reduce events greater than one cell per well. The latter events were subsequently discarded by inspection. The selective cloning medium, consisting of 50% conditioned medium obtained from CHO-MC1 cultures and 50% fresh RPMI, was enriched with 20% fetal calf serum, 4 mM L-glutamine and 0.2 mM essential amino acids without addition of IL-2. Cloning efficiency was determined by means of an analogous cloning experiment performed on untreated CTLL cells in the presence of IL-2.
ACKNOWLEDGEMENTS
Thanks are due to A. Grisanti for assistance with cell irradiation and to Luigina Renzi for helping with the photomicroscopy. We are grateful to Silvia Bacchetti and Davide Lazzareschi for stimulating discussions. This work was supported by grants 97.01096.PF49 and 97.01176.PF49 from the Consiglio Nazionale delle Ricerche and BIO2-CT94-3069 from the Commission of European Communities.
REFERENCES
*To whom correspondence should be addressed. Tel: +39 06 4991 7588; Fax: +39 06 4991 7594; Email: pdonini{at}axcasp.caspur.it
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