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25 Banerjee, S. et al. (1998) Am. Chem. Soc. Symp. Ser. 684, 125143 26 Akkara, J. A., Aranda, F. A., Rao, D. V. G. L. N. and John, V. T. (1998) in Electrical and Optical Polymer Systems (Vol. 13) (Wise, D. L. et al., eds), pp. 453465, Marcel Dekker 27 Shoda, S. and Kobayashi, S. (1997) Trends Polym. Sci. 5, 109115 28 Lee, J. H. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 74257429 29 Kobayashi, S., Wen, X. and Shoda, S. (1996) Macromolecules 29, 26982700 30 Kamat, S. V. et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 29402944 31 MacDonald, R. T. et al. (1995) Macromolecules 28, 7378 32 Uyama, H., Takeya, K. and Kobayashi, S. (1995) Bull. Chem. Soc. Jpn. 68, 5661 33 Kamat, S. V., Beckman, E. J. and Russell, A. J. (1993) J. Am. Chem. Soc. 115, 88458846 34 Russell, A. J., Beckman, E. J. and Chaudhary, A. K. (1994) CHEMTECH 24, 3337 35 Klibanov, A. M. (1990) Acc. Chem. Res. 23, 114116 36 Mayer, J. M. and Kaplan, D. L. (1994) Trends Polym. Sci. 2, 227230 37 Powers, M. E. et al. (1993) Biopolymer 33, 927932 38 Paradkar, V. M., Paradkar, M. and Dordick, J. S. (1994) J. Am. Chem. Soc. 116, 5009511

Comparative gene-expression analysis


Detlef H. Kozian and Bernhard J. Kirschbaum
The study of differences in gene-expression patterns is one of the most promising approaches for understanding mechanisms of differentiation and development. In addition, the identification of disease-related target molecules opens new avenues for rational pharmaceutical intervention. Recent technical advances and improvements are accelerating the analysis of gene-expression profiles at the transcript level. The knowledge and comprehension of currently applied methods is one of the central criteria for an efficient and successful gene-screening approach.

n the early years of the next millennium, the entire human genome will have been analysed through the Human Genome Project. The increasing amount of sequence data resulting from this endeavour provides ever more information about the composition of the genome, completing the search for pieces of the genomic puzzle. Comparisons with sequences from public databases already frequently allow the function of newly isolated genes to be predicted. Far more detailed functional studies on expression patterns of the putative gene and biochemical analysis of the corresponding protein must follow. Both classical and morerecent tools of molecular biology offer excellent ways to study the expression profiles of previously insufficiently characterized genes and to isolate new cDNA sequences. The addition of functional data to public databases will offer the possibility of characterizing genes in a more function-orientated way and may provide a rationale for subsequent studies. Currently, there are two main approaches to the analysis of molecular expression patterns: (1) the generation of mRNA-expression maps and (2) examination of the proteome, in which the expression profile of proteins is analysed by techniques such as two-dimensional gel electrophoresis, mass spectrometry [matrix-assisted-desorption-ionizationtime-offlight (MALDI-TOF) or electrospray] and by the ability to sequence subpicomole amounts of protein.

Classical approaches to transcript imaging, such as northern blotting or plaque hybridization, are timeconsuming and material-intensive ways to analyse mRNA-expression patterns. For these reasons, enormous effort has been undertaken to develop methods for high-throughput screening in industrial and clinical research. Currently, tools for studying geneexpression at the mRNA-level can be divided into three major groups, each being an excellent molecular tool for specific purposes (Box 1). This article is an overview of current methods for transcript imaging, with an emphasis on technologies that have been developed during the past few years that allow the highthroughput analysis of mRNA-expression profiles. Classical approaches A breakthrough in the analysis of gene expression was the development of the northern-blot technique in 19771 . With this technique, labelled cDNA or RNA probes are hybridized to RNA blots to study the expression patterns of mRNA transcripts. Even 20 years later, northern-blot analyses have not lost their importance in molecular biology; regardless of the technique initially used, differences detected in transcript expression are almost always confirmed by northern-blot analysis. Alternatively, RNase-protection assays can detect the expression of specific RNAs. Initially, these assays were mainly used to map RNAs2 but kits are now commercially available that allow the expression of mRNA subsets to be determined in a parallel manner. For RNaseprotection assays, the sequence of the analysed mRNA has to be known in order to synthesize a labelled cDNA

D. H. Kozian (detlef.kozian@hmrag.com) and B. J. Kirschbaum are at, Hoechst Marion Roussel Deutschland, The Center for Applied Genomics, Fraunhoferstrasse 22, 82152 Martinsried, Germany.
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Box 1. Current methods for the study of gene expression at the transcript level
Hybridization-based techniques Northern blotting S1-mapping/RNase protection Differential plaque hybridization Subtraction cloning DNA microarrays PCR-based techniques Differential display RDA (representational difference analysis) Sequence-based techniques ESTs (expressed sequence tags) SAGE (serial analysis of gene expression) MPSS (massively parallel signature sequencing) DNA-sequencing chip Mass-spectrometry sequencing

YXAAAAAAAAAAA-3 5-T11XY-3 dNTP Reverse transcriptase YXAAAAAAAAAAA-3 YXTTTTTTTTTTTTT-5 5-T11XY-3 5-ATGGTCTCAA-3 dNTP Taq Polymerase (35S) dATP

RT

5 3

PCR

5-ATGGTCTCAA 5-ATGGTCTCAA 5-ATGGTCTCAA 3

that forms a hybrid with the selected mRNA; such hybrids resist RNA degradation by a single-strand-specific nuclease and can be detected by gel electrophoresis. As a third approach, differential plaque-filter hybridization allows the identification of specific differences in the expression of cloned cDNAs3. One of the first experiments using this technique resulted in the identification and isolation of galactose-inducible yeast genes4. Since then, numerous new genes derived from different organisms have been isolated and cloned with this technique. Although all of these techniques are excellent tools for studying differences in gene expression, the limiting factor of these classical methods is that expression patterns can be analysed only for known genes. The analysis of gene-expression patterns made a significant advance with the development of subtractive cDNA libraries, which are generated by hybridizing an mRNA pool of one origin to an mRNA pool of a different origin. Transcripts that do not find a complementary strand in the hybridization step are then used for the construction of a cDNA library5. A variety of refinements to this method have been developed to identify specific mRNAs6,7. One of these is the selective amplification of differentially expressed mRNAs via biotin- and restriction-mediated enrichment (SABRE)8. cDNAs derived from a tester population are hybridized against the cDNAs of a driver (control) population. After a purification step specific for testercDNA-containing hybrids, testertester homohybrids are specifically amplified using an added linker, thus allowing the isolation of previously unknown genes. For the large-scale analysis of gene-expression at the mRNA level, new methods have been developed that allow the profiling of gene expression in a parallel fashion. To describe these methods in detail exceeds the scope of this review, but it is worthwhile discussing the rationale behind the development of these emerging technologies. Differential display When the technique of differential display of eukaryotic mRNA was first published in 1992, it was presented as a fast and reliable method for comparing differential gene-expression of two or more cell populations or tissues9. In fact, it was the first one-tube method to analyse and compare transcribed genes systematically in a bidirectional fashion; subtractive and differential hybridization techniques have only been adapted for the unidirectional identification of differentially expressed genes. To improve the performance of differential display, a battery of refinements were proposed to strengthen its reproducibility and efficiency10,11. Initially performed with radioactive nucleotides, the subsequent use of fluorescently labelled amplification primers provided safer working conditions12. A simplified standard protocol illustrates how differential display works (Fig. 1). The most critical point that should be considered when applying differential display is the source and purity of RNA. Poorly defined maintenance conditions for the cells or tissues might render further work difficult. DNase-treated total RNA of high purity is reverse transcribed using a T11XY primer (X A,C,G; Y A,C,G,T), which serves as the template for subsequent PCR. The PCR is performed using a radiolabelled nucleotide, the same
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YXTTTTTTTTTTT-5 Analysis of amplified cDNAs on a sequencing gel

Figure 1 Differential display of expressed mRNAs. Total mRNA is reverse transcribed (RT) and amplified with random ten-mer primers. The analysis of radioactive labelled PCR products in a sequencing gel gives information about the gene-expression status of the analysed cell or tissue. Differentially expressed genes (detectable as bands present in only one lane of the gel) are cut out of the gel and further analysed.

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T11XY primer used for the reverse transcription and a set of random decamer primers. Each of these primer sets will amplify a subset of all cDNAs, resulting in the generation of up to a hundred cDNA-fragments in one reaction tube. A portion of the PCR sample is then size fractionated with denaturing gel electrophoresis and the pattern of the amplified cDNAs is visualized autoradiographically. Comparisons of the cDNA band pattern lead to the identification of differentially amplified cDNAs, which can then be eluted from the gel, reamplified, cloned and sequenced. Differential display has been successfully applied in various areas, such as the detection and isolation of genes that are responsive to growth factors, developmentally regulated genes and genes whose expression correlates with certain disease states. The most significant advantages of differential display are its simplicity and the possibility of detecting virtually all expressed mRNAs when using sufficient primer combinations. The use of differential display is particularly advantageous in situations where the availability of RNA is limited, because very small amounts of total RNA are needed. Furthermore, it permits high-throughput screening of differentially amplified cDNAs and the rapid isolation and cloning of genes of interest. To minimise redundancy in the amplified cDNA sequences, a method was developed in 1996 to analyse 3 -end restriction fragments of differentially expressed cDNAs13. Reverse transcription is carried out using a T11XY with an additional priming site. After treating the cDNA pool with a restriction enzyme, a linker is added to the 5 end of the cDNA fragments to allow highstringency PCR for the generation of cDNAs without any redundancy. Only the extreme 3 ends of the cDNAs will be amplified, owing to the presence of a 3 priming site; all other cDNA fragments lack a priming site and are not amplified. The expression pattern of known sequences of a predicted size serves as an internal standard. Owing to the possibility of analysing the expression patterns of several hundred genes in a single experiment and the immediate availability of cDNA probes, differential RNA display is one of the most suitable methods for tracking novel genes, although the generation of false positives remains one of its major drawbacks. Representational difference analysis Originally developed to identify differences between two complex genomes, representational difference analysis (RDA) was adapted to analyse differential gene expression by taking advantage of both subtractive hybridization and PCR14,15. In the first step, mRNA derived from two different populations, the tester and the driver (control), is reverse transcribed (Fig. 2); the tester cDNA represents the cDNA population in which differential gene expression is expected to occur. Following digestion with a frequently cutting restriction endonuclease, linkers are ligated to both ends of the cDNA. A PCR step then generates the initial representation of the different gene pools. The linkers of the tester and driver cDNA are digested and a new linker is ligated to the ends of the tester cDNA. The tester and driver cDNAs are then mixed in a 1:100 ratio with an excess of driver cDNA in order to promote hybridization between single-stranded cDNAs common in both tester and driver cDNA pools. Following hybridization of the cDNAs, a PCR expoTIBTECH FEBRUARY 1999 (VOL 17)

AAAAAAAAA TTTTTTTTTT Cut tester and driver cDNA with a frequently cutting restriction endonuclease, ligation of linkers, fill in, PCR Tester Driver

Digest linker Ligate new linker

Digest linker

Hybridize in a 1:100 ratio

Only testertester hybrids are amplified


Figure 2 Schematic diagram of representational difference analysis (RDA). mRNA of different origins (tester and driver) is reverse transcribed, digested with a frequently cutting restriction endonuclease and then amplified via the ligated linker. The linkers of both cDNA pools are removed and a new linker is added to the tester cDNA. After hybridization of the tester and driver cDNAs and subsequent PCR, only those cDNAs present in the tester population are amplified.

nentially amplifies only those homoduplexes generated by the tester cDNA, via the priming sites on both ends of the double-stranded cDNA. The major advantage of RDA is the specific amplification of fragments exclusively present in one cDNA pool, owing to an enrichment of rarely expressed tester sequences16. The combination of subtractive hybridization and specific amplification of differentially expressed mRNAs provides target cDNA of high purity. To achieve an improved efficiency, alternating rounds of difference enrichment are required. Like differential mRNA display, RDA has been successfully applied to isolate differentially expressed genes during development, diseases and in growth-factor-stimulated cells1719. Considering their handling and efficiency, differential mRNA display and RDA are the most suitable methods for analysing differential gene-expression for small laboratories. Expressed sequence tags The gene-expression pattern of a cell or organism determines its basic biological characteristics. In microorganisms, the identification of putative coding regions can be achieved by sequencing the entire genome. For eukaryotic organisms, alternative approaches have been employed that take into account the noncontiguous organization of eukaryotic genes. In order to accelerate the discovery and characterization of mRNA-encoding sequences, the idea emerged to sequence fragments of cDNA randomly, direct from

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a variety of tissues20,21. This new paradigm rapidly filled the public and private gene banks with new sequence information and provides an individual fingerprint of the expression status of the analysed tissue or cell. One of the most important hallmarks of these expressed sequence tags (ESTs) is that they allow the identification of coding regions in genome-derived sequences. Overlapping EST sequences can be assembled into contigs, allowing the complete mRNA sequence of a gene to be determined. Considering that, in a couple of years, the sequencing of the human genome will be finished, the generation of databases with ESTs will help the expressed genomic regions to be distinguished from the non-expressed. A very promising feature of publicly available EST databases is the comparative analysis of gene expression in silico. As recently shown, differentially expressed genes can be identified by comparing the databases of expressed sequence tags of a given organ or cell type with sequence information from a different origin22,23. Sequencing of ESTs results in a huge amount of

sequence information and gives a profile of the geneexpression status. However, as large-scale sequencing facilities are required, it does not seem to be the right tool for medium-sized laboratories. Serial analysis of gene expression Serial analysis of gene expression (SAGE) is a sequence-based approach to the identification of differentially expressed genes through comparative analyses24. It allows the simultaneous analysis of sequences that derive from different cell populations or tissues. Three steps form the molecular basis for SAGE: (1) generation of a sequence tag (1014 bp) to identify expressed transcripts; (2) ligation of sequence tags to obtain concatemers that can be cloned and sequenced; and (3) comparison of the sequence data to determine differences in expression of genes that have been identified by the tags. To generate sequence tags, mRNA is reverse transcribed with a biotinylated oligo(dT) primer and then digested with a frequent-cutting restriction enzyme (the anchoring enzyme) (Fig. 3). The 3 portion of the cDNA is purified through binding to streptavidin-coated beads. This cDNA pool is split into two fractions (A and B), to each of which a primer (A and B ) that contains a recognition site for a type-IIS restriction enzyme is ligated (these restriction enzymes cleave DNA at a defined distance from their recognition site). After digestion with the relevant type-IIS restriction enzyme (the tagging enzyme) and elution of the digested and unbound DNA portion, the eluted DNA fragments are ligated and amplified using the primers A and B . Following PCR, the primer sites A and B are removed with the anchoring enzyme. The sticky ends thus generated enable the DNA fragments to form concatemers, which can then be cloned into a vector. The structure of the concatemers has a typical pattern: between each anchoring site, a so-called digitag contains the sequence information of two independent cDNA tags. This procedure is performed for every mRNA popuation to be analysed. Based on the sequence information, comparative computational analyses for the presence and the frequency of transcripts can be performed. It has been recently shown that the frequency of genes found in SAGE correlates strongly with their abundance in the corresponding cDNA libraries24. One might question whether short sequence tags (1014 bp) contain sufficient information to identify a transcript uniquely, given that the tag is obtained from a single position within each transcript. However, it has been shown that this is indeed the case. The major drawback of SAGE is undoubtedly the fact that corresponding genes can be identified only for those tags that are deposited in gene banks, thus making the efficiency of SAGE dependent on the complexity of available databases. As a sequence-based approach, virtually all transcripts present in a cell population or tissue can be tagged and analysed within a single experimental set-up. This technique has been applied successfully to the detection of p53-induced gene expression before the onset of apoptosis and to the analysis of the expression profiles of normal versus cancer cells25,26. When using automated sequencers and computational support, this technique allows rapid expression profiling of genes that are deposited in databases, thus making this technique suitable for average-sized laboratories.
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AE AE AE

AAAAAAAAA TTTTTTTTTTB AAAAAAAAA TTTTTTTTTTB AAAAAAAAA TTTTTTTTTTB

Cleave with anchoring enzyme (AE) Bind to streptavidin Separate sample in two aliquots Ligation of linkers A and B Linker ATECATG Linker ATEGTAC AAAAAAAAA TTTTTTTTTTB Linker BTECATG Linker BTEGTAC AAAAAAAAA TTTTTTTTTTB

Cleave with tagging enzyme (TE) Blunt end Ligation Amplification with primers A and B Linker A NNNNNCATGXXXXXXXXXOOOOOOOOOCATGNNNNN Linker B Linker A NNNNNGTACXXXXXXXXXOOOOOOOOOGTACNNNNN Linker B AE Digitag AE

Cleave with anchoring enzyme (AE) Ligation, clone into vector CATGXXXXXXXXXOOOOOOOOCATGXXXXXXXXOOOOOOOOCATG GTACXXXXXXXXXOOOOOOOOGTACXXXXXXXXOOOOOOOOGTAC

Sequencing of concatemers

Figure 3 Experimental procedure for the serial analysis of gene expression (SAGE). cDNA is digested with an anchoring enzyme (AE) and purified via the biotinylated oligo(dT) primer (B). A linker containing an internal tagging-enzyme site (TE) is ligated to the 5 ends of the cDNAs (anchoring enzyme and tagging enzyme are two different restriction endonucleases). The removal of the linker with a tagging enzyme and the ligation and amplification of the eluted DNA fragments leads to the generation of concatemers. These concatemers are composed of a number of digitags, which are sequence tags of two independent genes (X tag of the first gene, O tag of the second gene). Subsequent sequence analysis of the concatemers allows profiling of the gene expression.

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DNA microarrays A completely different approach to the study of geneexpression profiles and genome composition has been developed with the introduction of DNA microarrays. Current DNA microarrays are systematically gridded at high density. Such microarrays are generated by using cDNAs (e.g. ESTs), PCR products or cloned DNA, which are linked to the surface of nylon filters, glass slides or silicon chips27. DNA arrays can also be assembled from synthetic oligonucleotides, either by directly applying the synthesized oligonucleotides to the matrix or by a more sophisticated method that combines photolithography and solid-phase chemical synthesis28. To determine differences in gene-expression, labelled cDNAs or oligonucleotides are hybridized to the DNAor oligomer-carrying arrays. When using different fluorophores for labelling cDNAs or oligonucleotides, two probes can be applied simultaneously to the array and compared at different wavelengths. The expression of 10 000 genes and more can be analysed on a single chip29. The principal advantages of this approach are the ability to analyse expression patterns in a parallel fashion and the immediate interpretation of hybridization results. As oligonucleotides are much shorter probes than cDNAs, the hybridization stringency is weaker. The use of peptide nucleic acid (PNA) oligonucleotides may help to circumvent this problem because of the high stability of PNADNA duplexes30. If the oligonucleotides linked to the surface of a chip are long enough to provide specific hybridization events in concert with the photolithography technology for their synthesis, such arrays are excellent tools for high-throughput screenings. However, it must be emphasized that, for this technique, gene expression should always be measured in a dynamic range. Depending on the sensitivity of both cDNA and oligonucleotide arrays, the intensity of hybridization signals can leave the linear range when either weakly or abundantly expressed genes are analysed. Thus, individual optimization steps are required to ensure the accurate detection of differentially expressed genes. Outlook In the past few years, highly sophisticated tools have been developed for the analysis of differential gene expression. Each of these techniques has a number of unique advantages, such as simplicity (differential display) or the range (SAGE) of analysis. Conversely, there are also limitations, including the unidirectional analysis of RDA, the high expense of DNA microarrays or the analysis only of known genes with SAGE. Most of the discussed techniques have evolved from the need for a molecular tool for a specific application. Standard approaches such as northern-blot analysis, subtractive hybridization or differential plaque hybridization are labour and material intensive, unidirectional, serial or not well suited to the detection of rare mRNA species. However, they remain indispensable tools for sizing mRNAs and full-length cloning. The introduction of differential display provided a fast and technically simple method for the identification of differentially expressed mRNAs. This nonselective approach, which offers the possibility of comparing gene expression in a parallel and bidirectional fashion, is one of the most commonly used methods for gene-expression analysis.
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Sequence-based approaches such as SAGE or oligonucleotide arrays are excellent tools for high-throughput screenings of expression profiles but, once differentially expressed mRNAs are detected, the corresponding cDNAs must be cloned. Other promising sequencebased technologies such as massively parallel signature sequencing (MPSS), DNA-sequencing chips and massspectrometry sequencing remain to be evaluated by the scientific community. It is highly desirable to have an ideal tool, which would allow the unambiguous identification of differentially expressed genes in a simple and parallel manner and be suited to the identification of low-abundance mRNA species in a single experiment. Promising approaches in the field of protein analysis, proteomics, have the capacity to complement molecular approaches based on DNA- and RNA-expression studies. The proteome-analysis approach has the advantage of being closer to the biological consequences of altered gene expression. It benefits from the wealth of sequence information accumulated by genome-based approaches, which allow peptide data to be connected directly to nucleotide sequences and gene information. Although in the early stages of development, proteome research has already proved to offer indispensable molecular tools for extensive expression studies for organisms such as Saccharomyces cerevisiae, Escherichia coli or Haemophilus influenzae31. Molecular biotechnology is still progressing to develop new technologies that satisfy the demands of gene-expression analyses. For the time being, we will have to combine the currently available tools in an intelligent way. Based on experience, equipment and cost, one is left to decide which technology best suits the needs of the laboratory and project. It is the combination of classical and modern molecular tools that makes molecular-biological approaches so powerful for the discovery of new genes, helping to understand mechanisms of development and differentiation, as well as the genesis and progression of diseases at the molecular level. References
1 Alwine, J. C., Kemp, D. J. and Stark, G. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 53505354 2 Berk, A. J. and Sharp, P. A. (1977) Cell 12, 721732 3 Maniatis, T. et al. (1978) Cell 15, 687701 4 St John, T. P. and Davis, R. W. (1979) Cell 16, 443452 5 Hedrick, S. M., Cohen, D. I., Nielsen, E. A. and Davis, M. M. (1984) Nature 308, 149153 6 Swaroop, A., Xu, J. Z., Agarwal, N. and Weissman, S. M. (1991) Nucleic Acids Res. 25, 1954 7 Diatchenko, L. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 60256030 8 Lavery, D. J., Lopez-Molina, L., Fleury-Olela, F. and Schibler, U. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 68316836 9 Liang, P. and Pardee, A. B. (1992) Science 257, 967971 10 Bauer, D. et al. (1993) Nucleic Acids Res. 11, 42724280 11 Liang, P. and Pardee, A. B. (1995) Curr. Opin. Immunol. 7, 274280 12 Ito, T. and Sakaki, Y. (1997) Methods Mol. Biol. 85, 3744 13 Prashar, Y. and Weissman, S. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 659663 14 Lisitsyn, N., Lisitsyn, N. and Wigler, M. (1993) Science 259, 946951 15 Hubank, M. and Schatz, D. G. (1994) Nucleic Acids Res. 22, 56405648 16 ONeill, M. J. and Sinclair, A. (1997) Nucleic Acids Res. 25, 26812682 17 Gress, T. M. et al. (1997) Genes Chromosomes Cancer 19, 97103

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18 Wada, J., Kumar, A., Ota, K., Wallner, E. I., Batlle, D. C. and Kanwar, Y. S. (1997) Kidney Int. 51, 16291638 19 Edman, C. F., Prigent, S. A., Schipper, A. and Feramisco, J. R. (1997) Biochem. J. 323, 113118 20 Adams, M. D. et al. (1991) Science 252, 16511656 21 Adams, M. D. et al. (1995) Nature 377, 316 22 Lee, N. H. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 83038307 23 Vasmatzis, G., Essand, M., Brinkmann, U., Lee, B. and Pastan, I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 300304 24 Velculescu, V. E., Zhang, L., Vogelstein, B. and Kinzler, K. W. (1995) Science 270, 484487 25 Zhang, L. et al. (1997) Science 276, 12681272 26 Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W. and Vogelstein, B. (1997) Nature 389, 300305 27 Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. (1995) Science 270, 467470 28 Fodor, P. A., Rava, R. P., Huang, X. C., Pease, A. C., Holmes, C. P. and Adams, C. L. (1993) Nature 364, 555556 29 Chee, M. et al. (1996) Science 274, 610614 30 Hoheisel, J. D. (1997) Trends Biotechnol. 15, 465469 31 Humphrey-Smith, I., Stuart, S. J. and Blackstock, W. P. (1997) Electrophoresis 18, 12171242

The in vivo delivery of heterologous proteins by microencapsulated recombinant cells


Patricia L. Chang, Jeremy M. Van Raamsdonk, Gonzalo Hortelano, Susan C. Barsoum, Nicole C. MacDonald and Tracy L. Stockley
The microencapsulation of recombinant cells is a novel and potentially cost-effective method of heterologous protein delivery. A universal cell line, genetically modified to secrete any desired protein, is immunologically protected from tissue rejection by enclosure in microcapsules. The microcapsule can then be implanted in different recipients to deliver recombinant proteins in vivo.

he biotechnology industry is entering the new millennium at a particularly exciting stage. Recent advances in molecular biology have provided entirely new avenues for gene-based applications, especially in areas of health care, agriculture and animal husbandry. In addition to the classical pharmaceutical approach of synthesizing drugs chemically, genes are now used as templates and cells as reactors to provide the end products. By genetic engineering, these templates are introduced directly into cells in which they are expressed as novel proteins. In essence, expressing genes in vivo represents a cost-effective method of drug delivery, the drug in question being a gene product with a desired therapeutic or commercial value. Based on this concept, universal recombinant cell lines in immunoisolation devices have been developed as vehicles for drug delivery in vivo1. In this approach, cell lines are engineered to secrete a desired gene product. Enclosing these cells in immunoprotective devices would allow such nonautologous cells to be implanted into any host to deliver the desired gene product without triggering graft rejection. The primary requirement is that such

devices must have a permeability threshold that allows transit of the necessary nutrients and recombinant products, while entry of the hosts immune mediators is prohibited by their larger molecular sizes (Fig. 1). The advantages of this nonautologous method of gene delivery are: (1) it does not require chemical purification of the gene product, an often daunting task in terms of labor and cost; (2) it does not require modification of the hosts genome, thus providing additional measures of safety and cost saving; (3) it provides ample material for quality assessment before implantation, a safety feature not available to most other forms of in vivo delivery; and (4) the delivery of the recombinant product can be controlled so that it can be provided continuously, periodically (in response to extraneous signaling molecules such as tetracycline2 or other low molecular weight synthetic ligands3) or even shut off entirely, depending on the requirements of the condition being treated. Technical considerations Biomaterials The basic principle of microencapsulation is the protection of the encapsulated cells from the immune system while allowing the enclosed cells to survive and secrete a therapeutic product in a foreign host. The major role of the encapsulation material is to provide an immunoprotective environment for transplanted cells4,5, and must, in addition, be biocompatible and mechanically stable. Although numerous biomaterials fabricated as microcapsules or diffusion chambers have been studied for the purpose of immunoisolation
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P. L. Chang (changp@fhs.mcmaster.ca) and T. L. Stockley are at the Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada L8N 3Z5. J. M. Van Raamsdonk is at the Department of Medical Sciences, McMaster University. G. Hortelano is at the Department of Pathology, McMaster University. S. C. Barsoum and N. C. MacDonald are at the Department of Biology, McMaster University. G. Hortelano and T. L. Stockley are also at the Canadian Red Cross, Hamilton, Ontario, Canada.

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