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Evaluation of Phenotypic Alteration by Microcell-Mediated Chromosome Transfer
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
238, 107–116 (1996)
0263
REVIEW Evaluation of Phenotypic Alteration by MicrocellMediated Chromosome Transfer Jay D. Hunt1 Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center and Stanley S. Scott Cancer Center, New Orleans, Louisiana 70112
Genetic evaluation through somatic cell hybridization is a useful technique in any system containing a phenotype capable of being reversed or altered. In fact, the use of somatic cell hybridization to elucidate the effect of the genome of a normal cell on the phenotype of a malignant cell was the first demonstration that regulatory genes in normal cells controlled the growth of the hybrid cells (1–3). This powerful technique was limited however, because it involved the transfer of the entire genome of both cells to the somatic cell hybrid. Although laborious, data were obtained as to the chromosome in which regulatory genes were located, often through serial passage and random elimination of the chromosomes (4–7) or by the analysis of large numbers of phenotypically repressed clones (8, 9). In contrast, in some cases it was impossible to determine the chromosome that carried the regulatory gene (10, 11). Through the careful cataloging of chromosomal aberrations, investigators can often predict with a reasonable amount of certainty in which chromosome a particular gene responsible for a disorder is located. Deletions and other chromosomal abnormalities that result in the loss of function of a gene often indicate the position in the chromosome where tumor suppressor genes, genes responsible for genetic disorders, or other genetic susceptibility loci are located (12–16). Perhaps the most well characterized as a group are the genetic aberrations responsible for the formation of neoplasms. In 1986, Mitelman (17) reported that after examining 5345 karyotypes from tumors, only 77 different aberrations were observed as the sole change in at least two cancers of the same morphology [reviewed by Heim and Mitelman (18)]. The 77 abnormalities were limited to 1 Address correspondence to author at Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center and Stanley S. Scott Cancer Center, 1901 Perdido Street, P7-2, New Orleans, LA 70112. Fax: (504) 599-1014. E-mail: [email protected].
83 bands which represented 25% of the human genome and excluded the sex chromosomes. Therefore, these chromosomal aberrations likely represented the position of critically important genes in the genome, like tumor suppressor genes and oncogenes. Initially using karyotypes and later applying molecular loss-of-heterozygosity analyses, many tumor suppressor genes have been localized to specific areas of chromosomes (19– 24). The first tumor suppressor gene to be isolated was the retinoblastoma susceptibility locus. In fact, it was the frequency of deletions and abnormalities of chromosome 13 in patients with retinoblastoma that led investigators to suspect that the susceptibility gene for this disease was located in chromosome 13 (25). Retinoblastoma is a rare intraocular pediatric tumor of the retina that presents in two forms. The more common sporadic type is unilateral and monofocal. In contrast, the less common hereditary form is bilateral and multifocal. In 1971, Knudson postulated that mutations in a single recessive regulatory gene were responsible for the development of retinoblastoma (26). He suggested that individuals predisposed to the disease inherited a faulty gene from one parent and thus required only a single mutation in the homolog to develop retinoblastoma. However, those individuals with two normal genes required two somatic mutations and thus developed only one tumor in one eye (27). The gene was eventually identified (28) and cloned (29). Returning the cloned gene to a retinoblastoma cell line suppressed tumorigenicity (30). To test the hypothesis that genes responsible for genetic disorders are located at the position of consistent chromosomal abnormalities, a functional assay for the gene located in the candidate chromosome is needed. Short of actually having identified and isolated the candidate gene, monochromosome transfer could confirm the location of genetic susceptibility loci and aid in the isolation of these genes. The first successful transfer of 107
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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a single chromosome to a somatic cell was reported by Fournier and Ruddle (31). In this study, murine chromosomes were transferred to mouse, Chinese hamster, and human cells using microcell-mediated chromosome transfer resulting in somatic cell hybrids that maintained the chromosomes as functioning genetic elements. Selection of transferred chromosomes was based on biochemical makers that complemented deficiencies of hypoxanthine–guanine phosphoribosyltransferase (HGPRT,2 EC 2.4.2.8), adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), and thymidine kinase (TK, EC 2.7.1.75). This seminal paper established microcell-mediated chromosome transfer as a fundamental method for the validation of the presence of a genetic susceptibility locus in a particular chromosome. Since this report was published, an evergrowing number of studies have used microcell-mediated chromosome transfer to yield valuable information on the location of many critical genes (Table 1). Although predominantly used as a tool for the suppression of tumorigenicity by cancer biologists, this technique has great potential for the identification or confirmation of the presence in a chromosome of any gene that alters the phenotype of a cell in a measurable fashion. For instance, monochromosome transfer of chromosome 3 to a cell line with a mismatch repair deficiency confirmed that a functional hMLH1, located in chromosome 3 (32, 33), could restore genetic stability to the hybrid cell (34). Likewise, in the genetic disease ataxia-telangiectasia, monochromosome transfer of a normal chromosome 11 resulted in the correction of enhanced killing and radioresistant DNA synthesis of ataxia-telangiectasia cells by X rays (ataxia-telangiectasia cells demonstrate an atypical response to DNA damage induced by ionizing radiation, such as increased cell killing and diminished inhibition of DNA synthesis) (35). In addition, monochromosome transfer of human chromosome 2 to a hamster mutant cell line defective in double-strand break rejoining allowed for the substantial fragmentation of the chromosome and for the genetic ordering of chromosome 2 markers (36). A PROTOCOL FOR MICROCELL-MEDIATED CHROMOSOME TRANSFER
Several published protocols for the preparation and use of microcells are available (31, 37–42) and are contrasted here. In this procedure (diagrammed in Fig. 1), microcells are generated by first causing micronucleation in the donor cells by their prolonged treatment 2 Abbreviations used: HGPRT, hypoxanthine–guanine phosphoribosyltransferase; FISH, fluorescent in situ hybridization; BSA, bovine serum albumin; XMMCT, irradiation microcell-mediated chromosome transfer.
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with colcemid. Donor cells are often the L-cell murine fibrosarcoma derivative cell line A9 that contains one human chromosome ‘‘tagged’’ with the neomycin phosphotransferase gene which provides resistance to the neomycin analog G418. Alternatively, genetic selection with biochemical markers or other antibiotic resistance genes can also be used. Treatment of the cells with cytochalasin B followed by centrifugation results in enucleation and formation of microcells. Microcells are purified by filtration and fused to recipient cells using polyethylene glycol. Selection in antibiotics or in medium supplemented with biochemicals follows fusion. A protocol commonly used by us for microcell-mediated chromosome transfer was modified from the protocol of Dowdy and Stanbridge (41) and is detailed here. In this protocol, microcells are generated by growing donor cells to 80% confluency in six 25-cm2 tissue culture flasks (Fig. 2A). The importance of the type of flask used (Costar, Cambridge, MA; Cat. No. 3025) cannot be overstressed because less substantial flasks will break under the force of centrifugation (see below). The donor cells are arrested in mitosis by treatment with 50 ng/ ml colcemid (Gibco/BRL, Grand Island, NY) for 48– 72 h in standard growth medium which results in the formation of micronuclei (Fig. 2B). The tissue culture flasks are then filled to capacity with serum-free growth medium supplemented with 10 mg/ml cytochalasin B (Sigma Chemical Co., St. Louis, MO) and are incubated at 377C for 20 min. Stocks of cytochalasin B are typically made at 5 mg/ml in dimethyl sulfoxide and are aliquoted for storage at 0207C. Treatment of the cells with cytochalasin B causes rapid blebbing of the membrane (Fig. 2C) and is essential for enucleation by centrifugation. The tissue culture flasks are placed into a JA-10 (Beckman Instruments, Palo Alto, CA) rotor in which 150 ml of prewarmed water (377C) has been placed into each well of the rotor to serve as a cushion (note that we have also used a Beckman JA14 rotor with a 100-ml water cushion). Flasks are placed into the rotor so that the bottoms of the flasks are located at the bottom-outside of the rotor, and the tops of the flasks are oriented toward the center spindle of the rotor. Microcells are formed by centrifuging the cells attached to the tissue culture flask at 8000 rpm (maximum RCF 11,325g) for 65 min at 347C [for JA-14 rotor use 14,000 rpm (24,000g) for 65 min at 347C]. The relatively dense micronuclei migrate to the bottom of the tissue culture flask where a crude microcell pellet forms. After centrifugation, only spheroplasts remain attached to the surface of the flask (Fig. 2D). The medium is carefully poured from the tissue culture flasks as not to disturb the crude microcell pellet and can be reused for up to 2 weeks for future microcell generation if stored at 47C. The microcell crude preparation, which contains microcells, whole nuclei, and spheroplasts, is
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MONOCHROMOSOME TRANSFER TABLE 1
Genetic Disorders or Diseases Studied by Monochromosome Transfer Chromosome
Disorder or disease
Reference(s)
1 1 1 1 2 3 3 3 3 3 3 4 5 6 6 6 6 7 7 8 9 9 11 11 11 11 11 11 11 11 11 11 11 11 11 11 12 13 13 17 17 17 17 17 17 18 18
Human neuroblastoma Human colon carcinoma Human fibrosarcoma Human uterine endometrial carcinoma Human double-strand break repair gene Human ovarian carcinoma Murine fibrosarcoma Human renal cell carcinoma Mismatch repair deficiency and microsatellite instability Human bronchiolo-alveolar carcinoma Murine fibrosarcoma Human teratocarcinoma Human colorectal carcinoma Human breast carcinoma Metastatic human melanoma Human uterine endometrial carcinoma Human melanoma Murine squamous cell carcinoma Human immortalized fibroblasts Metastatic rat prostatic cancer Syrian hamster BHK cells Human uterine endometrial carcinoma Human breast carcinoma Human cutaneous squamous cell carcinoma Human neuroblastoma Human Wilms’ tumor Human ataxia-telangiectasia Human thyroid carcinoma Metastatic rat prostate cancer Human peripheral neuroepitheliomas Murine squamous cell carcinoma cell line Human cervical carcinoma Human fibrosarcoma Rhabdomyosarcoma Rat nephroblastoma Human bronchiolo-alveolar carcinoma Human prostate cancer Regulation of human retinoblastoma protein expression Human peripheral neuroepitheliomas Metastatic rat mammary cancer Metastatic rat prostate cancer Human breast cancer Human prostate cancer Human fibrosarcoma Human peripheral neuroepitheliomas Human endometrial carcinoma Human colorectal carcinoma
(54) (55) (56, 57) (58) (59) (60) (61) (62, 63) (34) (40) (64) (65) (66) (67) (68) (58) (69) (70) (71) (72) (73) (58) (67) (74) (54) (75, 76) (35) (77) (78) (79) (80) (81, 82) (57) (83) (83) (40) (84) (85) (79) (78) (78) (86, 87) (88) (89) (79) (90) (66)
placed into 100 ml of serum-free medium and is filtered sequentially through a 12-mm and then an 8-mm Nuclepore filter (Costar, 47-mm diameter attached to a 60-ml syringe) and twice through a 5-mm Nuclepore filter to remove whole cells and nuclei. The use of 47mm Swin-Lok filter holders (Costar) greatly facilitates the filtering process. It is important to allow the microcells to pass through the filters under the pull of gravity and without pressure from a syringe because accidental
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isolation of whole nuclei may occur. This process generally consumes about 1 h. The purified microcells are centrifuged in a clinical centrifuge at 2000 rpm for 10 min at 47C. The microcells are suspended in 6 ml of a lectin solution (50 mg/ml PHA-P, Sigma Chemical Co.) that promotes microcell adherence to recipient cells. Lectin stocks are typically made at 0.5 mg/ml in serumfree medium and are aliquoted for storage at 0207C. Before the microcell solution is placed on the recipient
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FIG. 1. Overview of microcell-mediated chromosome transfer procedure . The donor cell line, which contains the chromosome of interest, is arrested in mitosis by treatment with colcemid. Treatment of the cells with cytochalasin B followed by centrifugation results in the formation of a crude preparation of microcells. Microcells are isolated by filtration and are fused to recipient cells. Selection of hybrid cells precedes phenotypic evaluation.
cell line, the recipient cells, grown in six 60-mm tissue culture dishes, are washed three times in serum-free medium. The microcell solution (1 ml per 60-mm dish) is then incubated with the recipient cell line for 30 min at 377C to allow for the attachment of microcells to the recipient cells (Fig. 2E). After incubation, the solution is carefully aspirated from the plate, and cell fusion results from treatment with a freshly prepared 44% polyethylene glycol 1000 (Calbiochem, La Jolla, CA) solution (pH 7.5) for 1 min. The polyethylene glycol solution is washed from the plate with three gentle washes of serum-free medium. Polyethylene glycol is highly toxic to cells and should not be left on cells for more than 1 min. The cell hybrids are allowed to grow overnight and are then passed from the six 60-mm dishes to six 100-mm dishes in standard growth medium. After an overnight incubation to allow the hybrid cells to recover, the cells are selected with G418 (Gibco/ BRL) (or other selection medium as necessary). Colonies that grow in selection medium are isolated and cloned, and the presence of the transferred chromosome is confirmed by polymerase chain reaction-based restriction fragment-length polymorphism analyses, microsatellite analyses, or fluorescent in situ hybridization (FISH) analyses. The FISH analysis is usually composed of whole chromosome painting to determine if small regions of the transferred chromosome have been retained by the hybrid cell that might otherwise
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go undetected. Also, it is prudent to do appropriate analyses to determine if a whole or partial mouse chromosome has been transferred. This can be accomplished by FISH, Southern blot analysis, or sequence tag site (STS) mapping. After confirmation is made that the appropriate chromosome has been transferred, the phenotype of the hybrid cell is tested. As seen in Fig. 3, transfer of a single chromosome can have a profound affect on the phenotype of a cell line. In cancer studies, loss of tumorigenicity is usually measured by growth in semisolid medium or in immunocompromised or syngeneic animals. Several alternatives to this microcell method have been tried by us and are contrasted here where appropriate. Many procedures have included an incubation of the colcemid-treated cells in a hypotonic solution immediately prior to enucleation (37). We have found that this step can safely be omitted without detrimental affects on the production of microcells. Initially, we used the protocol of Sanford and Stubblefield (37) who generated microcells in suspension. In this method, microcells were generated by centrifugation of micronucleated cells in a Percoll (Pharmacia Biotech, Piscataway, NJ) gradient containing cytochalasin B. Isopyknic gradients were formed by centrifugation in a Beckman JA-20 rotor for 70 min at 377C at 19,000 rpm (maximum RCF 43,666g). After centrifugation, two bands were visible in the gradients and were removed using a sterile Pasteur pipet. Generally, this protocol yielded lower numbers of transferred chromosomes than the currently used method (detailed above). As an alternative to the purification of microcells by filtration, we have used unit gravity sedimentation on linear 1–3% bovine serum albumin (BSA) gradients (31). In this procedure, the crude preparation of microcells is suspended in 2 ml of 0.5% BSA in phosphatebuffered saline (PBS), which is layered onto 50 ml of the 1–3% BSA gradient. Separation is allowed to occur for 3.5 h at room temperature. The top 20 ml of the gradient was used. Again, we have found that the use of Nuclepore filters is far superior and results in less contamination, a faster purification, and higher transfer efficiencies. We have also used fusion in suspension as an alternative to fusion on monolayers (91). This method results in large numbers of somatic cell hybrids; however, the main disadvantage to the use of suspension fusion is that recipient cell 1 recipient cell hybrids will occur. In many instances this will not be problematic if anticipated when the selection protocol is designed. However, recipient cell 1 recipient cell 1 microcell hybrids will inevitably occur and will likely not be selected against. The affects of a tetraploid genotype may limit gene dose affects on the phenotype and should be considered if fusion in suspension is selected.
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FIG. 2. The cell line A9/3neo (A) contains a single human chromosome 3 tagged with the neomycin phosphotransferase gene. Treatment of the cells for 48 h with colcemid results in the formation of micronuclei (B). Rapid blebbing of the membrane results from treatment with cytochalasin B (C). After centrifugation of the flask, all that remains on the surface of the plastic are spheroplasts (D). The recipient bronchiolo-alveolar cell line A549 is shown with several microcells attached to their cell surface immediately prior to fusion (E).
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FIG. 3. The morphology of a poorly differentiated lung adenocarcinoma is shown in typical cell culture growth (A). Note the poor organization and lack of colony growth. (B) Following transfer of chromosome 3 to this cell line, tight colonies are observed reminiscent of a more differentiated state.
In most recent instances, the L-cell murine fibrosarcoma derivative cell line A9 has been used as a donor cell line. Most protocols (including the protocol presented in this review) have been perfected for use of this cell line. We have used the Chinese hamster lung cell line E36 (92), which is defective in HGPRT, for a microcell donor cell line. Good efficiency was obtained in these studies, indicating that perhaps any rodent cell line may function well using this protocol. Human cell lines and normal cell strains have also been used as donor lines. These human cell lines are transfected with an antibiotic resistance marker and are used as donors with A9 cells serving as recipients. In this fashion, a somatic cell hybrid is produced that contains one human chromosome in an A9 murine background. IRRADIATION MICROCELL-MEDIATED CHROMOSOME TRANSFER
The standard microcell-mediated chromosome transfer technique previously described is used to introduce whole chromosomes into recipient cells. This will confirm that the gene of interest is present in the transferred chromosome, but does not identify the region in the chromosome in which the gene is located. To transfer less than a whole chromosome, irradiation of chromosomes prior to transfer results in random fragmentation and deletion of whole regions. The technique, termed irradiation microcell-mediated chromosome transfer (XMMCT) (41, 43), is very similar to the protocol described in the previous section. In XMMCT, the enriched pure microcells are obtained by filtration with Nuclepore filters and then are suspended in 2 ml of serum-free growth medium supplemented with 50 mg/ ml PHA-P. The purified microcells are irradiated with 480 rads of g radiation using a 137Cs source. The micro-
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cells are then fused to the mouse cell line A9 and selected as above. After selection, resistant clones are isolated, karyotyped, and analyzed with marker loci to determine if deletions of the transferred chromosome have been obtained. It is sagacious to first map loss of loci on the transferred chromosome utilizing a PCRbased strategy using loci that are widely separated on the chromosome and then following this with more detailed mapping of clones which show interesting patterns of loss. A similar protocol based on XMMCT has been developed that results in subchromosomal transferable fragments (44) (Fig. 4). In this protocol, a selectable marker is transfected into a murine host somatic cell line that contains only one human chromosome of interest. The selectable marker integrates into all of the chromosomes randomly. Standard microcell-mediated chromosome transfer is then conducted to transfer the tagged chromosomes to murine A9 cells. Cells that result from the transfer of human chromosomes to the A9 cells are isolated. This can be facilitated by using a double selection strategy. For instance, if the chromosome of interest has previously been tagged with a different selectable marker or if the chromosome can be selected for by a biochemical marker, then a double selection scheme can be used (see Fig. 4). Microcells are generated from a pool of all cells that were transfected and have undergone double selection for the transfected marker (it is important to note that individual clones are not used and are not desirable for this procedure). The microcells are purified by filtration as described above. Microcells are then irradiated with 10,000 rads of g radiation resulting in the fragmentation of chromosomes in the microcells. Because pools of nonclonal cells were used to generate the microcells, there will
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FIG. 4. A general procedure for the development of subchromosomal transferable elements. In this example, a cell line that contains a human chromosome tagged with the hygromycin B resistance gene (HyB) is transfected with pSV2-neo which imparts resistance to the neomycin analog G418 (Neo). Microcells are generated from the transfected cells and fused with A9 recipient cells. The resultant HyBR and NeoR cells contain the human chromosome with varying integration sites of the Neo marker. Microcells are generated from polyclonal pools of the double-resistant cells, which are irradiated prior to fusion with A9 recipient cells. The resultant NeoR clones will contain fragments of the transferred human chromosome that all contain the Neo marker. Finally, the fragments can be transferred to recipient cells in order to evaluate alteration in a phenotype.
be many microcells containing subfragments of chromosomes with the transfected selectable marker integrated randomly throughout the chromosome of interest. Fusion of the irradiated microcells with the murine cell line A9 and supplemental selection of clones result in a large number of clones that contain fragments of the chromosome of interest. The resultant subchromosomal transferable elements are intermediate in size between yeast artificial chromosomes and whole chromosomes. This technique is particularly useful in dissociating complex phenotypic changes caused by the transfer of a whole chromosome. CONCLUSIONS
Clearly the field of somatic cell genetics has come a long way since the days of cell–cell fusion. The develop-
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ment of the microcell-mediated chromosome transfer technique has allowed for the less laborious examination of the effects of a single chromosome on the phenotype of a cell line without the need to detect phenotypic regression after random chromosomal elimination. Recent refinements to the technique, such as the development of irradiation microcell-mediated chromosome transfer and subchromosomal transferable elements, have further increased the utility of the assay. Two limitations to the use of these techniques have been the lack of available suitable hybrid panels with representation of each of the human chromosomes and the need for several different selection markers to allow for the transfer of two chromosomes to the same hybrid cell. Recently, two groups have constructed human:rodent monochromosome panels that contain each of the human autosomes tagged with the Hytk marker (45,
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46). The panel constructed by Cuthbert et al. (45) is the best characterized of the two and represents each of the 22 autosomes and the X chromosome. The Hytk marker codes for hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes in a bifunctional fusion sequence incorporated into a retroviral vector. Incubation of cells in medium supplemented with hygromycin B positively selects for the presence of the marker, and selection in medium supplemented with ganciclovir selects for loss of the Hytk gene. The use of the Hytk fusion gene as a selective genetic marker was insightful for two reasons: [1] it will allow for dual selection of more than one chromosome per somatic hybrid and [2] it will allow for the negative selection for the absence of the transferred chromosome so that a given phenotype observed with transfer of an individual chromosome can be corroborated by loss of the phenotype following reverse selection. This panel is freely available as a central human genetic resource (45). It is unlikely that all of the applications of monochromosome transfer have been explored. Because the field of molecular genetics is expanding so quickly, there is a constant need to update laborious methods. Positional cloning methods have traditionally been arduous. This is due in part to the use of unstable and cumbersome cloning vectors such as yeast artificial chromosomes (47). More stable vectors such as bacterial artificial chromosomes (48) and P1-derived bacterial artificial chromosomes (49) have been designed, but are small, making large cloning projects difficult. In addition, yeast artificial chromosomes, although transferable to mouse cells, are not routinely transferable to human cells (50–53). As panels of subchromosomal elements become available, their use may replace yeast artificial chromosomes as large cloning bodies because they can be transferred to and maintained in human cells. Finally, with technical improvements in the method, subchromosomal transferable elements may eventually be used as clinical gene therapy vectors. ACKNOWLEDGMENTS The author is supported in part by Louisiana Education Quality Support Fund Grant LEQSF(1995–1998)-RD-A-16, by a grant from the Cancer Association of Greater New Orleans, and by the Stanley S. Scott Cancer Center at LSUMC–New Orleans.
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62. Yoshida, M. A., Shimizu, M., Ikeuchi, T., Tonomura, A., Yokota, J., and Oshimura, M. (1994) Mol. Carcinog. 9, 114–121. 63. Shimizu, M., Yokota, J., Mori, N., Shuin, T., Shinoda, M., Terada, M., and Oshimura, M. (1990) Oncogene 5, 185–194. 64. Killary, A. M., Wolf, M. E., Giambernardi, T. A., and Naylor, S. L. (1992) Proc. Natl. Acad. Sci. USA 89, 10877–10881. 65. McGowan-Jordan, I. J., Speevak, M. D., Blakey, D., and Chevrette, M. (1994) Cancer Res. 54, 2568–2572. 66. Goyette, M. C., Cho, K., Fasching, C., Levy, D. B.,, Kinzler, K. W., Paraskeva, C., Vogelstein, B., and Stanbridge, E. J. (1992) Mol. Cell. Biol. 12, 1387–1395.
41. Dowdy, S. F., and Stanbridge, E. J. (1993) in Methods in Molecular Genetics: Gene and Chromosome Analysis Part A (Adolph, K. W., Ed.), pp. 151–160, Academic Press, San Diego.
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68. Welch, D. R., Chen, P., Miele, M. E., McGary, C. T., Bower, J. M., Stanbridge, E. J., and Weissman, B. E. (1994) Oncogene 9, 255–262. 69. Trent, J. M., Stanbridge, E. J., McBride, H. L., Meese, E. U., Casey, G., Araujo, D. E., Witkowski, C. M., and Nagle, R. B. (1990) Science 247, 568–571. 70. Zenklusen, J. C., Oshimura, M., Barrett, J. C., and Conti, C. J. (1994) Oncogene 9, 2817–2825. 71. Ogata, T., Ayusawa, D., Namba, M., Takahashi, E., Oshimura, M., and Oishi, M. (1993) Mol. Cell. Biol. 13, 6036–6043. 72. Ichikawa, T., Nihei, N., Suzuki, H., Oshimura, M., Emi, M., Nakamura, Y., Hayata, I., Isaacs, J. T., and Shimazaki, J. (1994) Cancer Res. 54, 2299–2302. 73. Islam, M. Q., Islam, K., Levan, G., and Horvath, G. (1995) Genes Chromosomes Cancer 13, 115–125. 74. Conway, K., Morgan, D., Phillips, K. K., Yuspa, S. H., and Weissman, B. E. (1992) Cancer Res. 52, 6487–6495. 75. Weissman, B. E., Saxon, P. J., Pasquale, S. R., Jones, G. R., Geiser, A. G., and Stanbridge, E. J. (1987) Science 236, 175– 180. 76. Dowdy, S. F., Fasching, C. L., Araujo, D., Lai, D.-M., Livanos, E., Weissman, B. E., and Stanbridge, E. J. (1991) Science 254, 293–295. 77. Yoshida, A., Asaga, T., Masuzawa, C., and Kawahara, S. (1994) J. Surg. Oncol. 55, 170–174.
53. Eliceiri, B., Labella, T., Hagino, Y., Srivastava, A., Schlessinger, D., Pilia, G., Palmieri, G., and D’Urso, M. (1991) Proc. Natl. Acad. Sci. USA 88, 2179–2183. 54. Bader, S. A., Fashing, C., Brodeur, G. M., and Stanbridge, E. J. (1991) Cell Growth Differ. 2, 245–255.
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0263
REVIEW Evaluation of Phenotypic Alteration by MicrocellMediated Chromosome Transfer Jay D. Hunt1 Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center and Stanley S. Scott Cancer Center, New Orleans, Louisiana 70112
Genetic evaluation through somatic cell hybridization is a useful technique in any system containing a phenotype capable of being reversed or altered. In fact, the use of somatic cell hybridization to elucidate the effect of the genome of a normal cell on the phenotype of a malignant cell was the first demonstration that regulatory genes in normal cells controlled the growth of the hybrid cells (1–3). This powerful technique was limited however, because it involved the transfer of the entire genome of both cells to the somatic cell hybrid. Although laborious, data were obtained as to the chromosome in which regulatory genes were located, often through serial passage and random elimination of the chromosomes (4–7) or by the analysis of large numbers of phenotypically repressed clones (8, 9). In contrast, in some cases it was impossible to determine the chromosome that carried the regulatory gene (10, 11). Through the careful cataloging of chromosomal aberrations, investigators can often predict with a reasonable amount of certainty in which chromosome a particular gene responsible for a disorder is located. Deletions and other chromosomal abnormalities that result in the loss of function of a gene often indicate the position in the chromosome where tumor suppressor genes, genes responsible for genetic disorders, or other genetic susceptibility loci are located (12–16). Perhaps the most well characterized as a group are the genetic aberrations responsible for the formation of neoplasms. In 1986, Mitelman (17) reported that after examining 5345 karyotypes from tumors, only 77 different aberrations were observed as the sole change in at least two cancers of the same morphology [reviewed by Heim and Mitelman (18)]. The 77 abnormalities were limited to 1 Address correspondence to author at Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center and Stanley S. Scott Cancer Center, 1901 Perdido Street, P7-2, New Orleans, LA 70112. Fax: (504) 599-1014. E-mail: [email protected].
83 bands which represented 25% of the human genome and excluded the sex chromosomes. Therefore, these chromosomal aberrations likely represented the position of critically important genes in the genome, like tumor suppressor genes and oncogenes. Initially using karyotypes and later applying molecular loss-of-heterozygosity analyses, many tumor suppressor genes have been localized to specific areas of chromosomes (19– 24). The first tumor suppressor gene to be isolated was the retinoblastoma susceptibility locus. In fact, it was the frequency of deletions and abnormalities of chromosome 13 in patients with retinoblastoma that led investigators to suspect that the susceptibility gene for this disease was located in chromosome 13 (25). Retinoblastoma is a rare intraocular pediatric tumor of the retina that presents in two forms. The more common sporadic type is unilateral and monofocal. In contrast, the less common hereditary form is bilateral and multifocal. In 1971, Knudson postulated that mutations in a single recessive regulatory gene were responsible for the development of retinoblastoma (26). He suggested that individuals predisposed to the disease inherited a faulty gene from one parent and thus required only a single mutation in the homolog to develop retinoblastoma. However, those individuals with two normal genes required two somatic mutations and thus developed only one tumor in one eye (27). The gene was eventually identified (28) and cloned (29). Returning the cloned gene to a retinoblastoma cell line suppressed tumorigenicity (30). To test the hypothesis that genes responsible for genetic disorders are located at the position of consistent chromosomal abnormalities, a functional assay for the gene located in the candidate chromosome is needed. Short of actually having identified and isolated the candidate gene, monochromosome transfer could confirm the location of genetic susceptibility loci and aid in the isolation of these genes. The first successful transfer of 107
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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a single chromosome to a somatic cell was reported by Fournier and Ruddle (31). In this study, murine chromosomes were transferred to mouse, Chinese hamster, and human cells using microcell-mediated chromosome transfer resulting in somatic cell hybrids that maintained the chromosomes as functioning genetic elements. Selection of transferred chromosomes was based on biochemical makers that complemented deficiencies of hypoxanthine–guanine phosphoribosyltransferase (HGPRT,2 EC 2.4.2.8), adenine phosphoribosyltransferase (APRT, EC 2.4.2.7), and thymidine kinase (TK, EC 2.7.1.75). This seminal paper established microcell-mediated chromosome transfer as a fundamental method for the validation of the presence of a genetic susceptibility locus in a particular chromosome. Since this report was published, an evergrowing number of studies have used microcell-mediated chromosome transfer to yield valuable information on the location of many critical genes (Table 1). Although predominantly used as a tool for the suppression of tumorigenicity by cancer biologists, this technique has great potential for the identification or confirmation of the presence in a chromosome of any gene that alters the phenotype of a cell in a measurable fashion. For instance, monochromosome transfer of chromosome 3 to a cell line with a mismatch repair deficiency confirmed that a functional hMLH1, located in chromosome 3 (32, 33), could restore genetic stability to the hybrid cell (34). Likewise, in the genetic disease ataxia-telangiectasia, monochromosome transfer of a normal chromosome 11 resulted in the correction of enhanced killing and radioresistant DNA synthesis of ataxia-telangiectasia cells by X rays (ataxia-telangiectasia cells demonstrate an atypical response to DNA damage induced by ionizing radiation, such as increased cell killing and diminished inhibition of DNA synthesis) (35). In addition, monochromosome transfer of human chromosome 2 to a hamster mutant cell line defective in double-strand break rejoining allowed for the substantial fragmentation of the chromosome and for the genetic ordering of chromosome 2 markers (36). A PROTOCOL FOR MICROCELL-MEDIATED CHROMOSOME TRANSFER
Several published protocols for the preparation and use of microcells are available (31, 37–42) and are contrasted here. In this procedure (diagrammed in Fig. 1), microcells are generated by first causing micronucleation in the donor cells by their prolonged treatment 2 Abbreviations used: HGPRT, hypoxanthine–guanine phosphoribosyltransferase; FISH, fluorescent in situ hybridization; BSA, bovine serum albumin; XMMCT, irradiation microcell-mediated chromosome transfer.
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with colcemid. Donor cells are often the L-cell murine fibrosarcoma derivative cell line A9 that contains one human chromosome ‘‘tagged’’ with the neomycin phosphotransferase gene which provides resistance to the neomycin analog G418. Alternatively, genetic selection with biochemical markers or other antibiotic resistance genes can also be used. Treatment of the cells with cytochalasin B followed by centrifugation results in enucleation and formation of microcells. Microcells are purified by filtration and fused to recipient cells using polyethylene glycol. Selection in antibiotics or in medium supplemented with biochemicals follows fusion. A protocol commonly used by us for microcell-mediated chromosome transfer was modified from the protocol of Dowdy and Stanbridge (41) and is detailed here. In this protocol, microcells are generated by growing donor cells to 80% confluency in six 25-cm2 tissue culture flasks (Fig. 2A). The importance of the type of flask used (Costar, Cambridge, MA; Cat. No. 3025) cannot be overstressed because less substantial flasks will break under the force of centrifugation (see below). The donor cells are arrested in mitosis by treatment with 50 ng/ ml colcemid (Gibco/BRL, Grand Island, NY) for 48– 72 h in standard growth medium which results in the formation of micronuclei (Fig. 2B). The tissue culture flasks are then filled to capacity with serum-free growth medium supplemented with 10 mg/ml cytochalasin B (Sigma Chemical Co., St. Louis, MO) and are incubated at 377C for 20 min. Stocks of cytochalasin B are typically made at 5 mg/ml in dimethyl sulfoxide and are aliquoted for storage at 0207C. Treatment of the cells with cytochalasin B causes rapid blebbing of the membrane (Fig. 2C) and is essential for enucleation by centrifugation. The tissue culture flasks are placed into a JA-10 (Beckman Instruments, Palo Alto, CA) rotor in which 150 ml of prewarmed water (377C) has been placed into each well of the rotor to serve as a cushion (note that we have also used a Beckman JA14 rotor with a 100-ml water cushion). Flasks are placed into the rotor so that the bottoms of the flasks are located at the bottom-outside of the rotor, and the tops of the flasks are oriented toward the center spindle of the rotor. Microcells are formed by centrifuging the cells attached to the tissue culture flask at 8000 rpm (maximum RCF 11,325g) for 65 min at 347C [for JA-14 rotor use 14,000 rpm (24,000g) for 65 min at 347C]. The relatively dense micronuclei migrate to the bottom of the tissue culture flask where a crude microcell pellet forms. After centrifugation, only spheroplasts remain attached to the surface of the flask (Fig. 2D). The medium is carefully poured from the tissue culture flasks as not to disturb the crude microcell pellet and can be reused for up to 2 weeks for future microcell generation if stored at 47C. The microcell crude preparation, which contains microcells, whole nuclei, and spheroplasts, is
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MONOCHROMOSOME TRANSFER TABLE 1
Genetic Disorders or Diseases Studied by Monochromosome Transfer Chromosome
Disorder or disease
Reference(s)
1 1 1 1 2 3 3 3 3 3 3 4 5 6 6 6 6 7 7 8 9 9 11 11 11 11 11 11 11 11 11 11 11 11 11 11 12 13 13 17 17 17 17 17 17 18 18
Human neuroblastoma Human colon carcinoma Human fibrosarcoma Human uterine endometrial carcinoma Human double-strand break repair gene Human ovarian carcinoma Murine fibrosarcoma Human renal cell carcinoma Mismatch repair deficiency and microsatellite instability Human bronchiolo-alveolar carcinoma Murine fibrosarcoma Human teratocarcinoma Human colorectal carcinoma Human breast carcinoma Metastatic human melanoma Human uterine endometrial carcinoma Human melanoma Murine squamous cell carcinoma Human immortalized fibroblasts Metastatic rat prostatic cancer Syrian hamster BHK cells Human uterine endometrial carcinoma Human breast carcinoma Human cutaneous squamous cell carcinoma Human neuroblastoma Human Wilms’ tumor Human ataxia-telangiectasia Human thyroid carcinoma Metastatic rat prostate cancer Human peripheral neuroepitheliomas Murine squamous cell carcinoma cell line Human cervical carcinoma Human fibrosarcoma Rhabdomyosarcoma Rat nephroblastoma Human bronchiolo-alveolar carcinoma Human prostate cancer Regulation of human retinoblastoma protein expression Human peripheral neuroepitheliomas Metastatic rat mammary cancer Metastatic rat prostate cancer Human breast cancer Human prostate cancer Human fibrosarcoma Human peripheral neuroepitheliomas Human endometrial carcinoma Human colorectal carcinoma
(54) (55) (56, 57) (58) (59) (60) (61) (62, 63) (34) (40) (64) (65) (66) (67) (68) (58) (69) (70) (71) (72) (73) (58) (67) (74) (54) (75, 76) (35) (77) (78) (79) (80) (81, 82) (57) (83) (83) (40) (84) (85) (79) (78) (78) (86, 87) (88) (89) (79) (90) (66)
placed into 100 ml of serum-free medium and is filtered sequentially through a 12-mm and then an 8-mm Nuclepore filter (Costar, 47-mm diameter attached to a 60-ml syringe) and twice through a 5-mm Nuclepore filter to remove whole cells and nuclei. The use of 47mm Swin-Lok filter holders (Costar) greatly facilitates the filtering process. It is important to allow the microcells to pass through the filters under the pull of gravity and without pressure from a syringe because accidental
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isolation of whole nuclei may occur. This process generally consumes about 1 h. The purified microcells are centrifuged in a clinical centrifuge at 2000 rpm for 10 min at 47C. The microcells are suspended in 6 ml of a lectin solution (50 mg/ml PHA-P, Sigma Chemical Co.) that promotes microcell adherence to recipient cells. Lectin stocks are typically made at 0.5 mg/ml in serumfree medium and are aliquoted for storage at 0207C. Before the microcell solution is placed on the recipient
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FIG. 1. Overview of microcell-mediated chromosome transfer procedure . The donor cell line, which contains the chromosome of interest, is arrested in mitosis by treatment with colcemid. Treatment of the cells with cytochalasin B followed by centrifugation results in the formation of a crude preparation of microcells. Microcells are isolated by filtration and are fused to recipient cells. Selection of hybrid cells precedes phenotypic evaluation.
cell line, the recipient cells, grown in six 60-mm tissue culture dishes, are washed three times in serum-free medium. The microcell solution (1 ml per 60-mm dish) is then incubated with the recipient cell line for 30 min at 377C to allow for the attachment of microcells to the recipient cells (Fig. 2E). After incubation, the solution is carefully aspirated from the plate, and cell fusion results from treatment with a freshly prepared 44% polyethylene glycol 1000 (Calbiochem, La Jolla, CA) solution (pH 7.5) for 1 min. The polyethylene glycol solution is washed from the plate with three gentle washes of serum-free medium. Polyethylene glycol is highly toxic to cells and should not be left on cells for more than 1 min. The cell hybrids are allowed to grow overnight and are then passed from the six 60-mm dishes to six 100-mm dishes in standard growth medium. After an overnight incubation to allow the hybrid cells to recover, the cells are selected with G418 (Gibco/ BRL) (or other selection medium as necessary). Colonies that grow in selection medium are isolated and cloned, and the presence of the transferred chromosome is confirmed by polymerase chain reaction-based restriction fragment-length polymorphism analyses, microsatellite analyses, or fluorescent in situ hybridization (FISH) analyses. The FISH analysis is usually composed of whole chromosome painting to determine if small regions of the transferred chromosome have been retained by the hybrid cell that might otherwise
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go undetected. Also, it is prudent to do appropriate analyses to determine if a whole or partial mouse chromosome has been transferred. This can be accomplished by FISH, Southern blot analysis, or sequence tag site (STS) mapping. After confirmation is made that the appropriate chromosome has been transferred, the phenotype of the hybrid cell is tested. As seen in Fig. 3, transfer of a single chromosome can have a profound affect on the phenotype of a cell line. In cancer studies, loss of tumorigenicity is usually measured by growth in semisolid medium or in immunocompromised or syngeneic animals. Several alternatives to this microcell method have been tried by us and are contrasted here where appropriate. Many procedures have included an incubation of the colcemid-treated cells in a hypotonic solution immediately prior to enucleation (37). We have found that this step can safely be omitted without detrimental affects on the production of microcells. Initially, we used the protocol of Sanford and Stubblefield (37) who generated microcells in suspension. In this method, microcells were generated by centrifugation of micronucleated cells in a Percoll (Pharmacia Biotech, Piscataway, NJ) gradient containing cytochalasin B. Isopyknic gradients were formed by centrifugation in a Beckman JA-20 rotor for 70 min at 377C at 19,000 rpm (maximum RCF 43,666g). After centrifugation, two bands were visible in the gradients and were removed using a sterile Pasteur pipet. Generally, this protocol yielded lower numbers of transferred chromosomes than the currently used method (detailed above). As an alternative to the purification of microcells by filtration, we have used unit gravity sedimentation on linear 1–3% bovine serum albumin (BSA) gradients (31). In this procedure, the crude preparation of microcells is suspended in 2 ml of 0.5% BSA in phosphatebuffered saline (PBS), which is layered onto 50 ml of the 1–3% BSA gradient. Separation is allowed to occur for 3.5 h at room temperature. The top 20 ml of the gradient was used. Again, we have found that the use of Nuclepore filters is far superior and results in less contamination, a faster purification, and higher transfer efficiencies. We have also used fusion in suspension as an alternative to fusion on monolayers (91). This method results in large numbers of somatic cell hybrids; however, the main disadvantage to the use of suspension fusion is that recipient cell 1 recipient cell hybrids will occur. In many instances this will not be problematic if anticipated when the selection protocol is designed. However, recipient cell 1 recipient cell 1 microcell hybrids will inevitably occur and will likely not be selected against. The affects of a tetraploid genotype may limit gene dose affects on the phenotype and should be considered if fusion in suspension is selected.
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FIG. 2. The cell line A9/3neo (A) contains a single human chromosome 3 tagged with the neomycin phosphotransferase gene. Treatment of the cells for 48 h with colcemid results in the formation of micronuclei (B). Rapid blebbing of the membrane results from treatment with cytochalasin B (C). After centrifugation of the flask, all that remains on the surface of the plastic are spheroplasts (D). The recipient bronchiolo-alveolar cell line A549 is shown with several microcells attached to their cell surface immediately prior to fusion (E).
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FIG. 3. The morphology of a poorly differentiated lung adenocarcinoma is shown in typical cell culture growth (A). Note the poor organization and lack of colony growth. (B) Following transfer of chromosome 3 to this cell line, tight colonies are observed reminiscent of a more differentiated state.
In most recent instances, the L-cell murine fibrosarcoma derivative cell line A9 has been used as a donor cell line. Most protocols (including the protocol presented in this review) have been perfected for use of this cell line. We have used the Chinese hamster lung cell line E36 (92), which is defective in HGPRT, for a microcell donor cell line. Good efficiency was obtained in these studies, indicating that perhaps any rodent cell line may function well using this protocol. Human cell lines and normal cell strains have also been used as donor lines. These human cell lines are transfected with an antibiotic resistance marker and are used as donors with A9 cells serving as recipients. In this fashion, a somatic cell hybrid is produced that contains one human chromosome in an A9 murine background. IRRADIATION MICROCELL-MEDIATED CHROMOSOME TRANSFER
The standard microcell-mediated chromosome transfer technique previously described is used to introduce whole chromosomes into recipient cells. This will confirm that the gene of interest is present in the transferred chromosome, but does not identify the region in the chromosome in which the gene is located. To transfer less than a whole chromosome, irradiation of chromosomes prior to transfer results in random fragmentation and deletion of whole regions. The technique, termed irradiation microcell-mediated chromosome transfer (XMMCT) (41, 43), is very similar to the protocol described in the previous section. In XMMCT, the enriched pure microcells are obtained by filtration with Nuclepore filters and then are suspended in 2 ml of serum-free growth medium supplemented with 50 mg/ ml PHA-P. The purified microcells are irradiated with 480 rads of g radiation using a 137Cs source. The micro-
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cells are then fused to the mouse cell line A9 and selected as above. After selection, resistant clones are isolated, karyotyped, and analyzed with marker loci to determine if deletions of the transferred chromosome have been obtained. It is sagacious to first map loss of loci on the transferred chromosome utilizing a PCRbased strategy using loci that are widely separated on the chromosome and then following this with more detailed mapping of clones which show interesting patterns of loss. A similar protocol based on XMMCT has been developed that results in subchromosomal transferable fragments (44) (Fig. 4). In this protocol, a selectable marker is transfected into a murine host somatic cell line that contains only one human chromosome of interest. The selectable marker integrates into all of the chromosomes randomly. Standard microcell-mediated chromosome transfer is then conducted to transfer the tagged chromosomes to murine A9 cells. Cells that result from the transfer of human chromosomes to the A9 cells are isolated. This can be facilitated by using a double selection strategy. For instance, if the chromosome of interest has previously been tagged with a different selectable marker or if the chromosome can be selected for by a biochemical marker, then a double selection scheme can be used (see Fig. 4). Microcells are generated from a pool of all cells that were transfected and have undergone double selection for the transfected marker (it is important to note that individual clones are not used and are not desirable for this procedure). The microcells are purified by filtration as described above. Microcells are then irradiated with 10,000 rads of g radiation resulting in the fragmentation of chromosomes in the microcells. Because pools of nonclonal cells were used to generate the microcells, there will
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FIG. 4. A general procedure for the development of subchromosomal transferable elements. In this example, a cell line that contains a human chromosome tagged with the hygromycin B resistance gene (HyB) is transfected with pSV2-neo which imparts resistance to the neomycin analog G418 (Neo). Microcells are generated from the transfected cells and fused with A9 recipient cells. The resultant HyBR and NeoR cells contain the human chromosome with varying integration sites of the Neo marker. Microcells are generated from polyclonal pools of the double-resistant cells, which are irradiated prior to fusion with A9 recipient cells. The resultant NeoR clones will contain fragments of the transferred human chromosome that all contain the Neo marker. Finally, the fragments can be transferred to recipient cells in order to evaluate alteration in a phenotype.
be many microcells containing subfragments of chromosomes with the transfected selectable marker integrated randomly throughout the chromosome of interest. Fusion of the irradiated microcells with the murine cell line A9 and supplemental selection of clones result in a large number of clones that contain fragments of the chromosome of interest. The resultant subchromosomal transferable elements are intermediate in size between yeast artificial chromosomes and whole chromosomes. This technique is particularly useful in dissociating complex phenotypic changes caused by the transfer of a whole chromosome. CONCLUSIONS
Clearly the field of somatic cell genetics has come a long way since the days of cell–cell fusion. The develop-
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ment of the microcell-mediated chromosome transfer technique has allowed for the less laborious examination of the effects of a single chromosome on the phenotype of a cell line without the need to detect phenotypic regression after random chromosomal elimination. Recent refinements to the technique, such as the development of irradiation microcell-mediated chromosome transfer and subchromosomal transferable elements, have further increased the utility of the assay. Two limitations to the use of these techniques have been the lack of available suitable hybrid panels with representation of each of the human chromosomes and the need for several different selection markers to allow for the transfer of two chromosomes to the same hybrid cell. Recently, two groups have constructed human:rodent monochromosome panels that contain each of the human autosomes tagged with the Hytk marker (45,
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46). The panel constructed by Cuthbert et al. (45) is the best characterized of the two and represents each of the 22 autosomes and the X chromosome. The Hytk marker codes for hygromycin phosphotransferase and herpes simplex virus thymidine kinase genes in a bifunctional fusion sequence incorporated into a retroviral vector. Incubation of cells in medium supplemented with hygromycin B positively selects for the presence of the marker, and selection in medium supplemented with ganciclovir selects for loss of the Hytk gene. The use of the Hytk fusion gene as a selective genetic marker was insightful for two reasons: [1] it will allow for dual selection of more than one chromosome per somatic hybrid and [2] it will allow for the negative selection for the absence of the transferred chromosome so that a given phenotype observed with transfer of an individual chromosome can be corroborated by loss of the phenotype following reverse selection. This panel is freely available as a central human genetic resource (45). It is unlikely that all of the applications of monochromosome transfer have been explored. Because the field of molecular genetics is expanding so quickly, there is a constant need to update laborious methods. Positional cloning methods have traditionally been arduous. This is due in part to the use of unstable and cumbersome cloning vectors such as yeast artificial chromosomes (47). More stable vectors such as bacterial artificial chromosomes (48) and P1-derived bacterial artificial chromosomes (49) have been designed, but are small, making large cloning projects difficult. In addition, yeast artificial chromosomes, although transferable to mouse cells, are not routinely transferable to human cells (50–53). As panels of subchromosomal elements become available, their use may replace yeast artificial chromosomes as large cloning bodies because they can be transferred to and maintained in human cells. Finally, with technical improvements in the method, subchromosomal transferable elements may eventually be used as clinical gene therapy vectors. ACKNOWLEDGMENTS The author is supported in part by Louisiana Education Quality Support Fund Grant LEQSF(1995–1998)-RD-A-16, by a grant from the Cancer Association of Greater New Orleans, and by the Stanley S. Scott Cancer Center at LSUMC–New Orleans.
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